Device and system for collecting and analyzing vapor condensate, particularly exhaled breath condensate, as well as method of using the same

ABSTRACT

The present invention is related to the field of bio/chemical sensing, assays and applications. Particularly, the present invention is related to collecting a small amount of a vapor condensate sample (e.g. the exhaled breath condensate (EBC) from a subject of a volume as small as 10 fL (femto-Liter) in a single drop), preventing or significantly reducing an evaporation of the collected vapor condensate sample, analyzing the sample, analyzing the sample by mobile-phone, and performing such collection and analysis by a person without any professionals.

CROSS-REFERENCING

This application claims the benefit of provisional application serialnos. 62/218,455 filed on Sep. 14, 2015, 62/293,188, filed on Feb. 9,2016, 62/305,123, filed on Mar. 8, 2016, 62/369,181, filed on Jul. 31,2016 and of PCT application serial no. PCT/US16/46437, filed on Aug. 20,2016, which PCT application claims the benefit of provisionalapplication serial nos. 62/202,989, filed on Aug. 10, 2015, 62/218,455filed on Sep. 14, 2015, 62/293,188, filed on Feb. 9, 2016, 62/305,123,filed on Mar. 8, 2016, and 62/369,181, filed on Jul. 31, 2016, all ofwhich applications are incorporated herein in their entireties for allpurposes.

FIELD

The present invention is related to the field of bio/chemical sampling,sensing, assays and applications.

BACKGROUND

In bio/chemical vapor condensate sample analysis, particularly exhaledbreath condensate (EBC), there is a need for the methods and devicesthat can simplify the sample collection and measurement processes, thatcan accelerate the process (e.g. binding, mixing reagents, etc.) andquantify the parameters (e.g. analyte concentration, the sample volume,etc.), that can handle samples with small volume, that allow an entireassay performed in less than a minute, that allow an assay performed bya smartphone (e.g. mobile phone), that allow non-professional to performan assay her/himself, and that allow a test result to be communicatedlocally, remotely, or wirelessly to different relevant parties. Thepresent invention relates to the methods, devices, and systems that canaddress these needs.

SUMMARY OF INVENTION

The following brief summary is not intended to include all features andaspects of the present invention. The present invention is related tothe field of bio/chemical sensing, assays and applications.Particularly, the present invention is related to collecting a smallamount of a vapor condensate sample (e.g. the exhaled breath condensate(EBC) from a subject of a volume as small as 10 fL (femto-Liter) in asingle drop), preventing or significantly reducing an evaporation of thecollected vapor condensate sample, analyzing the sample, analyzing thesample by mobile-phone, and performing such collection and analysis by aperson without any professionals.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way. The drawings maynot be in scale. In the figures that present experimental data points,the lines that connect the data points are for guiding a viewing of thedata only and have no other means.

FIG. 1 An illustration of certain aspects of an exemplary device andmethods of collecting exhaled breath condensate (EBC) using a SiEBCA(Single-drop EBC Collector/Analyzer).

FIG. 2 An illustration of different formations of EBC at closedconfiguration of SiEBCA depends on spacer height. In closedconfiguration-1: If spacer height is smaller than average height of EBCat open configuration; at closed configuration, EBC become a continuousthin film contacting both collection and cover plates and may have airisolated pockets. In the closed configuration-2: If spacer height islarger than average height of EBC at open configuration; at closedconfiguration EBC become isolated puddle(s) that contact both collectionand cover plates, and that are larger but fewer than that at the openconfiguration.

FIG. 3. An illustration of an embodiment of the devices and the methodsof a SiEBCA (Single-drop EBC Collector/Analyzer).

FIG. 4. An illustration of a SiEBCA with both “open spacer” and“enclosed spacer”, where the open spacer is a post (pillar) while theenclosed spacer is a ring spacer (d) and a four chamber grid spacer (e).

FIG. 5. The surface wetting properties for an untreated and a treated(for better wetting than untreated surface) surface of a collectionplate.

FIG. 6. Methods of pressing the plates of SiEBCA by human hand.

FIG. 7. Experimental data of EBC Droplets sizes and density on thecollection plate (untreated PMMA film) at an “open configuration” (e.g.only the collection plate without the cover plate.

FIG. 8. Experimental data of EBC formation on the collection plate whichis a surface treated PMMA film) at a plate open configuration.

FIG. 9. Photographs and measured evaporation time (at plate openconfiguration) of the EBC (2 s breathing directly from a subject)collected on untreated and treated PMMA plate.

FIG. 10. Photographs of spacer height effects (1 um, 2 um, 10 um and 30um, respectively) on the EBC collected using SiEBCA at the closedconfiguration.

FIG. 11. Experimental Data of Photographs of spacer height effects (1um, 2 um, 10 um and 30 um, respectively) on the EBC collected usingSiEBCA at the closed configuration.

FIG. 12. Photographs of the breath collected using the collection platethat are treated and untreated PMMA plates.

FIG. 13. Experimental data on effects of (a) treated and untreated PMMAcollection plates and (b) time delay in closing the cover plate onbreath collection.

FIG. 14. Experimental data of the volume of the collected breath (i.e.EBC) on the collection plate vs. the time delay (measured from the endof the breath to the covering of the cover plate) for the case of thetreated (which is more hydrophilic that the untreated) and untreatedcollection plate (PMMA) surface, respectively.

FIG. 15 shows reducing binding or mixing time by reducing the samplethickness using two plates, spacers, and compression (shown incross-section). Panel (a) illustrates reducing the time for bindingentities in a sample to a binding site on a solid surface (X−(Volume toSurface)). Panel (b) illustrates reducing the time for binding entities(e.g. reagent) stored on a surface of plate to a binding site on asurface of another surface (X−(Surface to Surface)). Panel (c)illustrates reducing the time for adding reagents stored on a surface ofa plate into a sample that is sandwiched between the plate and otherplate (X−(Surface to Volume)).

FIG. 16 schematically illustrates an exemplary embodiment of the presentinvention, a multiplexed detection in a single CROF device using onebinding site one plate and a plurality of storage sites on the otherplate. Panel (a) and (b) is a perspective and a cross-sectional view ofan exemplary device, respectively.

FIG. 17 schematically illustrates a further exemplary embodiment of thepresent invention, a multiplexed detection in a single CROF device usingone storage site on one plate and multiple binding sites on the otherplate. Panel (a) and (b) is a perspective and a cross-sectional view ofan exemplary device, respectively.

FIG. 18 schematically illustrates a further exemplary embodiment of thepresent invention, a multiplexed detection in a single CROF device withmultiple binding sites on one plate and multiple corresponding storagesites on another plate. Panel (a) and (b) is a perspective and across-sectional view of an exemplary device, respectively.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description illustrates some embodiments of theinvention by way of example and not by way of limitation. The sectionheadings and any subtitles used herein are for organizational purposesonly and are not to be construed as limiting the subject matterdescribed in any way. The contents under a section heading and/orsubtitle are not limited to the section heading and/or subtitle, butapply to the entire description of the present invention.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can need to be independentlyconfirmed.

The present invention is related to the field of bio/chemical sensing,assays and applications. Particularly, the present invention is relatedto collecting a small amount of a vapor condensate sample (e.g. theexhaled breath condensate (EBC) from a subject of a volume as small as10 fL (femto-Liter) in a single drop), preventing or significantlyreducing an evaporation of the collected vapor condensate sample,analyzing the sample, analyzing the sample by mobile-phone, andperforming such collection and analysis by a person without anyprofessionals. Since the exhaled breath condensate (EBC) and other vaporcondensate share many common properties, the disclosure uses EBC as arepresentative to illustrate certain embodiments of the presentinvention, but such presentation should not be construed as anylimitations of the present invention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence” and“oligonucleotide” are used interchangeably, and can also include pluralsof each respectively depending on the context in which the terms areutilized.

The term “capture agent” as used herein, refers to a binding member,e.g. nucleic acid molecule, polypeptide molecule, or any other moleculeor compound, that can specifically bind to its binding partner, e.g., asecond nucleic acid molecule containing nucleotide sequencescomplementary to a first nucleic acid molecule, an antibody thatspecifically recognizes an antigen, an antigen specifically recognizedby an antibody, a nucleic acid aptamer that can specifically bind to atarget molecule, etc.

The term “a secondary capture agent” which can also be referred to as a“detection agent” refers a group of biomolecules or chemical compoundsthat have highly specific affinity to the antigen. The secondary captureagent can be strongly linked to an optical detectable label, e.g.,enzyme, fluorescence label, or can itself be detected by anotherdetection agent that is linked to an optical detectable label throughbioconjugation (Hermanson, “Bioconjugate Techniques” Academic Press, 2ndEd., 2008).

The term “capture agent-reactive group” refers to a moiety of chemicalfunction in a molecule that is reactive with capture agents, i.e., canreact with a moiety (e.g., a hydroxyl, sulfhydryl, carboxyl or aminegroup) in a capture agent to produce a stable strong, e.g., covalentbond.

The terms “specific binding” and “selective binding” refer to theability of a capture agent to preferentially bind to a particular targetanalyte that is present in a heterogeneous mixture of different targetanalytes. A specific or selective binding interaction will discriminatebetween desirable (e.g., active) and undesirable (e.g., inactive) targetanalytes in a sample, typically more than about 10 to 100-fold or more(e.g., more than about 1000- or 10,000-fold).

The term “analyte” refers to a molecule (e.g., a protein, peptides, DNA,RNA, nucleic acid, or other molecule), cells, tissues, viruses, andnanoparticles with different shapes.

The term “assaying” refers to testing a sample to detect the presenceand/or abundance of an analyte.

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

As used herein, the term “light-emitting label” refers to a label thatcan emit light when under an external excitation. This can beluminescence. Fluorescent labels (which include dye molecules or quantumdots), and luminescent labels (e.g., electro- or chemi-luminescentlabels) are types of light-emitting label. The external excitation islight (photons) for fluorescence, electrical current forelectroluminescence and chemical reaction for chemi-luminescence. Anexternal excitation can be a combination of the above.

The phrase “labeled analyte” refers to an analyte that is detectablylabeled with a light emitting label such that the analyte can bedetected by assessing the presence of the label. A labeled analyte maybe labeled directly (i.e., the analyte itself may be directly conjugatedto a label, e.g., via a strong bond, e.g., a covalent or non-covalentbond), or a labeled analyte may be labeled indirectly (i.e., the analyteis bound by a secondary capture agent that is directly labeled).

The terms “hybridizing” and “binding”, with respect to nucleic acids,are used interchangeably.

The term “capture agent/analyte complex” is a complex that results fromthe specific binding of a capture agent with an analyte. A capture agentand an analyte for the capture agent will usually specifically bind toeach other under “specific binding conditions” or “conditions suitablefor specific binding”, where such conditions are those conditions (interms of salt concentration, pH, detergent, protein concentration,temperature, etc.) which allow for binding to occur between captureagents and analytes to bind in solution. Such conditions, particularlywith respect to antibodies and their antigens and nucleic acidhybridization are well known in the art (see, e.g., Harlow and Lane(Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1989) and Ausubel, et al, Short Protocols inMolecular Biology, 5th ed., Wiley & Sons, 2002).

A subject may be any human or non-human animal. A subject may be aperson performing the instant method, a patient, a customer in a testingcenter, etc.

As used herein, a “diagnostic sample” refers to any biological samplethat is a bodily byproduct, such as bodily fluids, that has been derivedfrom a subject. The diagnostic sample may be obtained directly from thesubject in the form of liquid, or may be derived from the subject byfirst placing the bodily byproduct in a solution, such as a buffer.Exemplary diagnostic samples include, but are not limited to, saliva,serum, blood, sputum, urine, sweat, lacrima, semen, feces, breath,biopsies, mucus, etc.

As used herein, an “environmental sample” refers to any sample that isobtained from the environment. An environmental sample may includeliquid samples from a river, lake, pond, ocean, glaciers, icebergs,rain, snow, sewage, reservoirs, tap water, drinking water, etc.; solidsamples from soil, compost, sand, rocks, concrete, wood, brick, sewage,etc.; and gaseous samples from the air, underwater heat vents,industrial exhaust, vehicular exhaust, etc. Typically, samples that arenot in liquid form are converted to liquid form before analyzing thesample with the present method.

As used herein, a “foodstuff sample” refers to any sample that issuitable for animal consumption, e.g., human consumption. A foodstuffsample may include raw ingredients, cooked food, plant and animalsources of food, preprocessed food as well as partially or fullyprocessed food, etc. Typically, samples that are not in liquid form areconverted to liquid form before analyzing the sample with the presentmethod.

The term “diagnostic,” as used herein, refers to the use of a method oran analyte for identifying, predicting the outcome of and/or predictingtreatment response of a disease or condition of interest. A diagnosismay include predicting the likelihood of or a predisposition to having adisease or condition, estimating the severity of a disease or condition,determining the risk of progression in a disease or condition, assessingthe clinical response to a treatment, and/or predicting the response totreatment.

A “biomarker,” as used herein, is any molecule or compound that is foundin a sample of interest and that is known to be diagnostic of orassociated with the presence of or a predisposition to a disease orcondition of interest in the subject from which the sample is derived.Biomarkers include, but are not limited to, polypeptides or a complexthereof (e.g., antigen, antibody), nucleic acids (e.g., DNA, miRNA,mRNA), drug metabolites, lipids, carbohydrates, hormones, vitamins,etc., that are known to be associated with a disease or condition ofinterest.

A “condition” as used herein with respect to diagnosing a healthcondition, refers to a physiological state of mind or body that isdistinguishable from other physiological states. A health condition maynot be diagnosed as a disease in some cases. Exemplary health conditionsof interest include, but are not limited to, nutritional health; aging;exposure to environmental toxins, pesticides, herbicides, synthetichormone analogs; pregnancy; menopause; andropause; sleep; stress;prediabetes; exercise; fatigue; chemical balance; etc.

The term “entity” refers to, but not limited to proteins, peptides, DNA,RNA, nucleic acid, molecules (small or large), cells, tissues, viruses,nanoparticles with different shapes, that would bind to a “bindingsite”. The entity includes the capture agent, detection agent, andblocking agent. The “entity” includes the “analyte”, and the two termsare used interchangeably.

The term “binding site” refers to a location on a solid surface that canimmobilize an entity in a sample.

The term “entity partners” refers to, but not limited to proteins,peptides, DNA, RNA, nucleic acid, molecules (small or large), cells,tissues, viruses, nanoparticles with different shapes, that are on a“binding site” and would bind to the entity. The entity, include, butnot limited to, capture agents, detection agents, secondary detectionagents, or “capture agent/analyte complex”.

The term “smart phone” or “mobile phone”, which are usedinterchangeably, refers to the type of phones that has a camera andcommunication hardware and software that can take an image using thecamera, manipulate the image taken by the camera, and communicate datato a remote place. In some embodiments, the Smart Phone has a flashlight.

The term “average linear dimension” of an area is defined as a lengththat equals to the area times 4 then divided by the perimeter of thearea. For example, the area is a rectangle, that has width w, and lengthL, then the average of the linear dimension of the rectangle is4*W*L/(2*(L+W)) (where “*” means multiply and “/” means divide). By thisdefinition, the average line dimension is, respectively, W for a squareof a width W, and d for a circle with a diameter d. The area include,but not limited to, the area of a binding site or a storage site.

The term “period” of periodic structure array refers to the distancefrom the center of a structure to the center of the nearest neighboringidentical structure.

The term “storage site” refers to a site of an area on a plate, whereinthe site contains reagents to be added into a sample, and the reagentsare capable of being dissolving into the sample that is in contract withthe reagents and diffusing in the sample.

The term “relevant” means that it is relevant to detection of analytes,quantification and/or control of analyte or entity in a sample or on aplate, or quantification or control of reagent to be added to a sampleor a plate.

The term “hydrophilic”, “wetting”, or “wet” of a surface means that thecontact angle of a sample on the surface is less than 90 degree.

The term “hydrophobic”, “non-wetting”, or “does not wet” of a surfacemeans that the contact angle of a sample on the surface is equal to orlarger than 90 degree.

The term “variation” of a quantity refers to the difference between theactual value and the desired value or the average of the quantity. Andthe term “relative variation” of a quantity refers to the ratio of thevariation to the desired value or the average of the quantity. Forexample, if the desired value of a quantity is Q and the actual value is(Q+□), the n the ⊏□/(Q+□) is the relative variation. The term “relativesample thickness variation” refers to the ratio of the sample thicknessvariation to the average sample thickness.

The term “optical transparent” refers to a material that allows atransmission of an optical signal, wherein the term “optical signal”refers to, unless specified otherwise, the optical signal that is usedto probe a property of the sample, the plate, the spacers, thescale-marks, any structures used, or any combinations of thereof.

The term “none-sample-volume” refers to, at a closed configuration of aCROF process, the volume between the plates that is occupied not by thesample but by other objects that are not the sample. The objectsinclude, but not limited to, spacers, air bubbles, dusts, or anycombinations of thereof. Often none-sample-volume(s) is mixed inside thesample.

The term “saturation incubation time” refers to the time needed for thebinding between two types of molecules (e.g. capture agents andanalytes) to reach an equilibrium. For a surface immobilization assay,the “saturation incubation time” refers the time needed for the bindingbetween the target analyte (entity) in the sample and the binding siteon plate surface reaches an equilibrium, namely, the time after whichthe average number of the target molecules (the entity) captured andimmobilized by the binding site is statistically nearly constant.

In some cases, the “analyte” and “binding entity” and “entity” areinterchangeable.

The term “first plate” and “collection plate are interchangeable. Theterm “second plate” and “cover plate” are interchangle.

A “processor,” “communication device,” “mobile device,” refer tocomputer systems that contain basic electronic elements (including oneor more of a memory, input-output interface, central processing unit,instructions, network interface, power source, etc.) to performcomputational tasks. The computer system may be a general purposecomputer that contains instructions to perform a specific task, or maybe a special-purpose computer.

A “site” or “location” as used in describing signal or datacommunication refers to the local area in which a device or subjectresides. A site may refer to a room within a building structure, such asa hospital, or a smaller geographically defined area within a largergeographically defined area. A remote site or remote location, withreference to a first site that is remote from a second site, is a firstsite that is physically separated from the second site by distanceand/or by physical obstruction. The remote site may be a first site thatis in a separate room from the second site in a building structure, afirst site that is in a different building structure from the secondsite, a first site that is in a different city from the second site,etc.

As used herein, the term “sample collection site” refers to a locationat which a sample may be obtained from a subject. A sample collectionsite may be, for example, a retailer location (e.g., a chain store,pharmacy, supermarket, or department store), a provider office, aphysician's office, a hospital, the subject's home, a military site, anemployer site, or other site or combination of sites. As used herein,the term “sample collection site” may also refer to a proprietor orrepresentative of a business, service, or institution located at, oraffiliated with, the site.

As used herein, “raw data” includes signals and direct read-outs fromsensors, cameras, and other components and instruments which detect ormeasure properties or characteristics of a sample.

“Process management,” as used herein, refers to any number of methodsand systems for planning and/or monitoring the performance of a process,such as a sample analysis process

One with skill in the art will appreciate that the present invention isnot limited in its application to the details of construction, thearrangements of components, category selections, weightings,pre-determined signal limits, or the steps set forth in the descriptionor drawings herein. The invention is capable of other embodiments and ofbeing practiced or being carried out in many different ways.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise, e.g., when the word “single” isused. For example, reference to “an analyte” includes a single analyteand multiple analytes, reference to “a capture agent” includes a singlecapture agent and multiple capture agents, reference to “a detectionagent” includes a single detection agent and multiple detection agents,and reference to “an agent” includes a single agent and multiple agents.

Device and System for Collecting and Analyzing Vapor Condensate,Particularly Exhaled Breath Condensate, as Well Method of Using the Same

Provided herein is advice for collecting and analyzing vapor condensate(VC) sample, comprising:

-   -   a collection plate and a cover plate, wherein:        -   i. the plates are movable relative to each other into            different configurations;        -   ii. one or both plates are flexible;        -   iii. each of the plates has, on its respective surface, a            sample contact area for contacting a vapor condensate (VC)            sample that contains an analyte;        -   iv. one or both of the plates comprise spacers that are            fixed with a respective plate, wherein the spacers have a            predetermined substantially uniform height and a            predetermined constant inter-spacer distance and wherein at            least one of the spacers is inside the sample contact area;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either completely or partially        separated apart, the spacing between the plates is not regulated        by the spacers, and the VC sample is deposited on one or both of        the plates; and    -   wherein another of the configurations is a closed configuration        which is configured after the VC sample deposition in the open        configuration; and in the closed configuration: at least a part        of the VC sample is between the two plates and in contact with        the two plates, and has a highly uniform thickness that is        regulated by the spacers and the two sample surfaces of the        plates and is equal to or less than 30 □m with a small        variation.

In some embodiments, the device further comprises, on one or bothplates, one or a plurality of dry binding sites and/or one or aplurality of reagent sites. In some embodiments, the sample is exhalebreath condensate.

In some embodiments, the sample is a vapor from a biological sample, anenvironmental sample, a chemical sample, or clinical sample. In someembodiments, wherein the analyte comprises a molecule (e.g., a protein,peptides, DNA, RNA, nucleic acid, or other molecules), cells, tissues,viruses, and nanoparticles with different shapes. In some embodiments,wherein the analyte comprises volatile organic compounds (VOCs). In someembodiments, wherein the analyte comprises nitrogen, oxygen, CO2, H2O,and inert gases. In some embodiments, wherein the analyte is stained.

In some embodiments, the device may comprise a dry reagent coated on oneor both of the plates. In some embodiments, the dry reagent may bind toan analyte in the blood an immobilize the analyte on a surface on one orboth of the plates. In these embodiments, the reagent may be an antibodyor other specific binding agent, for example. This dry reagent may havea pre-determined area. In other embodiments, the device may comprise areleasable dry reagent on one or more of the plates, e.g., a labeledreagent such as a cell stain or a labeled detection agent such as anantibody or the like. In some cases, there may be a release time controlmaterial on the plate that contains the releasable dry reagent, whereinthe release time control material delays the time that the releasabledry regent is released into the blood sample. In some cases, the releasetime control material delays the time that the dry regent starts isreleased into the blood sample by at least 3 seconds, e.g., at least 5seconds or at least 10 seconds. Some embodiments, the drive may containmultiple dry binding sites and/or multiple reagent sites, therebyallowing multiplex assays to be performed. In some cases, the areasoccupied by the drying binding sites may oppose the areas occupied bythe reagent sites when the plates are in the closed position.

In Some Embodiments, the Regent Comprises Labeling or StaniingReagent(s).

In some embodiments, the spacers regulating the layer of uniformthickness (i.e., the spacers that are spacing the plates away from eachother in the layer) have a “filling factor” of at least 1%, e.g., atleast 2% or at least 5%, wherein the filling factor is the ratio of thespacer area that is in contact with the layer of uniform thickness tothe total plate area that is in contact with the layer of uniformthickness. In some embodiments, for spacers regulating the layer ofuniform thickness, the Young's modulus of the spacers times the fillingfactor of the spacers is equal or larger than 10 MPa, e.g., at least 15MPa or at least 20 MPa, where the filling factor is the ratio of thespacer area that is in contact with the layer of uniform thickness tothe total plate area that is in contact with the layer of uniformthickness. In some embodiments, the thickness of the flexible platetimes the Young's modulus of the flexible plate is in the range 60 to750 GPa-um, e.g., 100 to 300 GPa-um, 300 to 550 GPa-um, or 550 to 750GPa-um. In some embodiments, for a flexible plate, the fourth power ofthe inter-spacer-distance (ISD) divided by the thickness of the flexibleplate (h) and the Young's modulus (E) of the flexible plate, ISD⁴/(hE),is equal to or less than 10⁶ um³/GPa, e.g., less than 10⁵ um³/GPa, lessthen 10⁴ um³/GPa or less than 10³ um³/GPa.

In some embodiments, one or both plates comprises a location markereither on a surface of or inside the plate, that provide information ofa location of the plate, e.g., a location that is going to be analyzedor a location onto which the blood should be deposited. In some cases,one or both plates may comprise a scale marker, either on a surface ofor inside the plate, that provides information of a lateral dimension ofa structure of the blood sample and/or the plate. In some embodiments,one or both plates comprises an imaging marker, either on surface of orinside the plate that assists an imaging of the sample. For example, theimaging marker could help focus the imaging device or direct the imagingdevice to a location on the device. In some embodiments, the spacers canfunction as a location marker, a scale marker, an imaging marker, or anycombination of thereof.

In some embodiments, on one of the sample surface, it further comprisesan enclosure-spacer that encloses a partial or entire VC samplesdeposited on the collection plate.

In some embodiments, the highly uniform thickness has a value equal toor less than 0.5 um. In some embodiments, the highly uniform thicknesshas a value in the range of 0.5 um to 1 um, 1 um to 2 um, 2 um to 10 um,10 um to 20 um or 20 um to 30 um.

In some embodiments, the thickness of the at least a part of VC sampleat the closed configuration is larger than the thickness of VC sampledeposited on the collection plate at an open configuration.

In some embodiments, the thickness of the at least a part of VC sampleat the closed configuration is less than the thickness of VC sampledeposited on the collection plate at an open configuration.

In some embodiments, wherein the spacing are fixed on a plate bydirectly embossing the plate or injection molding of the plate.

In some embodiments, wherein the materials of the plate and the spacersare selected from polystyrene, PMMA, PC, COC, COP, or another plastic.

In some embodiments, the inter-spacer spacing in the range of 1 um to 50um, 50 um to 100 um, 100 um to 200 um or 200 um to 1000 um.

In some embodiments, the VC sample is an exhaled breath condensate froma human or an animal.

In some embodiments, the spacers regulating the layer of uniformthickness have a filling factor of at least 1%, wherein the fillingfactor is the ratio of the spacer area in contact with the layer ofuniform thickness to the total plate area in contact with the layer ofuniform thickness.

In some embodiments, for spacers regulating the layer of uniformthickness, the Young's modulus of the spacers times the filling factorof the spacers is equal or larger than 10 MPa, wherein the fillingfactor is the ratio of the spacer area in contact with the layer ofuniform thickness to the total plate area in contact with the layer ofuniform thickness.

In some embodiments, for a flexible plate, the thickness of the flexibleplate times the Young's modulus of the flexible plate is in the range 60to 750 GPa-um.

In some embodiments, for a flexible plate, the fourth power of theinter-spacer-distance (ISD) divided by the thickness of the flexibleplate (h) and the Young's modulus (E) of the flexible plate, ISD⁴/(hE),is equal to or less than 10⁶ um³/GPa,

In some embodiments, one or both plates comprises a location marker,either on a surface of or inside the plate, that provide information ofa location of the plate.

In some embodiments, one or both plates comprises a scale marker, eitheron a surface of or inside the plate, that provide information of alateral dimension of a structure of the sample and/or the plate.

In some embodiments, one or both plates comprises an imaging marker,either on surface of or inside the plate, that assists an imaging of thesample.

In some embodiments, the spacers functions as a location marker, a scalemarker, an imaging marker, or any combination of thereof.

In some embodiments, the average thickness of the layer of uniformthickness is about equal to a minimum dimension of an analyte in thesample.

In some embodiments, the inter-spacer distance is in the range of 1 □m50 to 50 □m □m to 120 □m or 120 □m to 200 □n.

In some embodiments, the inter-spacer distance is substantiallyperiodic.

In some embodiments, the spacers are pillars with a cross-sectionalshape selected from round, polygonal, circular, square, rectangular,oval, elliptical, or any combination of the same.

In some embodiments, the spacers have are pillar shape and have asubstantially flat top surface, wherein, for each spacer, the ratio ofthe lateral dimension of the spacer to its height is at least 1.

In some embodiments, each spacer has the ratio of the lateral dimensionof the spacer to its height is at least 1.

In some embodiments, the minimum lateral dimension of spacer is lessthan or substantially equal to the minimum dimension of an analyte inthe sample.

In some embodiments, the minimum lateral dimension of spacer is in therange of 0.5 um to 100 um.

In some embodiments, the minimum lateral dimension of spacer is in therange of 0.5 um to 10 um.

In some embodiments, the spacers have a density of at least 100/mm².

In some embodiments, the spacers have a density of at least 1000/mm².

In some embodiments, at least one of the plates is transparent.

In some embodiments, at least one of the plates is made from a flexiblepolymer.

In some embodiments, for a pressure that compresses the plates, thespacers are not compressible and/or, independently, only one of theplates is flexible.

In some embodiments, the flexible plate has a thickness in the range of10 □m to 200 □m (e.g. about 10 um, 25 um, 50 um, 75 um, 100 um, 125 um,150 um, 175 um).

In some embodiments, the variation is less than 30%, 10%, 5%, 3% or 1%.

In some embodiments, the first and second plates are connected and areconfigured to be changed from the open configuration to the closedconfiguration by folding the plates.

In some embodiments, the first and second plates are connected by ahinge and are configured to be changed from the open configuration tothe closed configuration by folding the plates along the hinge.

In some embodiments, the first and second plates are connected by ahinge that is a separate material to the plates, and are configured tobe changed from the open configuration to the closed configuration byfolding the plates along the hinge

In some embodiments, the first and second plates are made in a singlepiece of material and are configured to be changed from the openconfiguration to the closed configuration by folding the plates.

In some embodiments, the layer of uniform thickness sample is uniformover a lateral area that is at least 100 um².

In some embodiments, the layer of uniform thickness sample is uniformover a lateral area that is at least 1 mm².

In some embodiments, the device is configured to analyze the sample in60 seconds or less.

In some embodiments, at the closed configuration, the final samplethickness device is configured to analyze the sample in 60 seconds orless.

In some embodiments, the device further comprises, on one or both of theplates, one or a plurality of amplification sites that are each capableof amplifying a signal from the analyte or a label of the analyte whenthe analyte or label is within 500 nm from an amplification site.

In some embodiments, at the closed configuration, the final samplethickness device is configured to analyze the sample in 10 seconds orless.

In some embodiments, the dry binding site comprises a capture agent.

In some embodiments, the dry binding site comprises an antibody ornucleic acid. In some embodiments, the releasable dry reagent is alabeled reagent. In some embodiments, the releasable dry reagent is afluorescently-labeled reagent. In some embodiments, the releasable dryreagent is a dye. In some embodiments, the releasable dry reagent is abeads. In some embodiments, the releasable dry reagent is a quantum dot.In some embodiments, the releasable dry reagent is afluorescently-labeled antibody.

In some embodiments, the first plate further comprises, on its surface,a first predetermined assay site and a second predetermined assay site,wherein the distance between the edges of the assay site issubstantially larger than the thickness of the uniform thickness layerwhen the plates are in the closed position, wherein at least a part ofthe uniform thickness layer is over the predetermined assay sites, andwherein the sample has one or a plurality of analytes that are capableof diffusing in the sample.

In some embodiments, the first plate has, on its surface, at least threeanalyte assay sites, and the distance between the edges of any twoneighboring assay sites is substantially larger than the thickness ofthe uniform thickness layer when the plates are in the closed position,wherein at least a part of the uniform thickness layer is over the assaysites, and wherein the sample has one or a plurality of analytes thatare capable of diffusing in the sample.

In some embodiments, the first plate has, on its surface, at least twoneighboring analyte assay sites that are not separated by a distancethat is substantially larger than the thickness of the uniform thicknesslayer when the plates are in the closed position, wherein at least apart of the uniform thickness layer is over the assay sites, and whereinthe sample has one or a plurality of analytes that are capable ofdiffusing in the sample.

In some embodiments, the releasable dry reagent is a cell stain. In someembodiments, the device further comprises a detector that is an opticaldetector for detecting an optical signal.

In some embodiments, the device further comprises a detector that is anelectrical detector for detecting an electric signal.

A system for rapidly analyzing a vapor condensation sample using amobile phone comprising:

-   -   (a) a device of any prior claim;    -   (b) a mobile communication device comprising:        -   i. one or a plurality of cameras for the detecting and/or            imaging the vapor condensate sample; and        -   ii. electronics, signal processors, hardware and software            for receiving and/or processing the detected signal and/or            the image of the vapor condensate sample and for remote            communication.

In some embodiments, the system further comprise a light source fromeither the mobile communication device or an external source.

In some embodiments, one of the plates has a binding site that binds ananalyte, wherein at least part of the uniform sample thickness layer isover the binding site, and is substantially less than the averagelateral linear dimension of the binding site.

In some embodiments, further comprising:

-   -   (d) a housing configured to hold the sample and to be mounted to        the mobile communication device.

In some embodiments, the housing comprises optics for facilitating theimaging and/or signal processing of the sample by the mobilecommunication device, and a mount configured to hold the optics on themobile communication device.

In some embodiments, an element of the optics in the housing is movablerelative to the housing.

In some embodiments, the mobile communication device is configured tocommunicate test results to a medical professional, a medical facilityor an insurance company.

In some embodiments, the mobile communication device is furtherconfigured to communicate information on the test and the subject withthe medical professional, medical facility or insurance company.

In some embodiments, the mobile communication device is furtherconfigured to communicate information of the test to a cloud network,and the cloud network process the information to refine the testresults.

In some embodiments, the mobile communication device is furtherconfigured to communicate information of the test and the subject to acloud network, the cloud network process the information to refine thetest results, and the refined test results will send back the subject.

In some embodiments, the mobile communication device is configured toreceive a prescription, diagnosis or a recommendation from a medicalprofessional.

In some embodiments, the mobile communication device is configured withhardware and software to:

-   -   a. capture an image of the sample;    -   b. analyze a test location and a control location in in image;        and    -   c. compare a value obtained from analysis of the test location        to a threshold value that characterizes the rapid diagnostic        test.

In some embodiments, at least one of the plates comprises a storage sitein which assay reagents are stored. In some embodiments, at least one ofthe cameras reads a signal from the CROF device. In some embodiments,the mobile communication device communicates with the remote locationvia a wifi or cellular network.

In some embodiments, the mobile communication device is a mobile phone.

A method for rapidly analyzing an analyte in a sample using a mobilephone, comprising:

-   -   a) depositing a sample on the device of any prior system claim;    -   b) assaying an analyte in the sample deposited on the device to        generate a result; and    -   c) communicating the result from the mobile communication device        to a location remote from the mobile communication device.

In some embodiments, the analyte comprises a molecule (e.g., a protein,peptides, DNA, RNA, nucleic acid, or other molecule), cells, tissues,viruses, and nanoparticles with different shapes.

In some embodiments, the analyte comprises white blood cell, red bloodcell and platelets.

In some embodiments, the method comprises:

-   -   a. analyzing the results at the remote location to provide an        analyzed result; and    -   b. communicating the analyzed result from the remote location to        the mobile communication device.

In some embodiments, the analysis is done by a medical professional at aremote location. In some embodiments, the mobile communication devicereceives a prescription, diagnosis or a recommendation from a medicalprofessional at a remote location.

In some embodiments, the thickness of the at least a part of VC sampleat the closed configuration is larger than the thickness of VC sampledeposited on the collection plate at an open configuration.

In some embodiments, the thickness of the at least a part of VC sampleat the closed configuration is less than the thickness of VC sampledeposited on the collection plate at an open configuration.

In some embodiments, the assaying step comprises detecting an analyte inthe sample.

In some embodiments, the analyte is a biomarker. In some embodiments,the analyte is a protein, nucleic acid, cell, or metabolite. In someembodiments, the assay done in step (b) is a binding assay or abiochemical assay.

A method for analyzing an analyte in a vapor condensate samplecomprising:

-   -   obtaining a device of any prior device claim;    -   depositing the vapor condensate sample onto one or both plates        of the device;    -   placing the plates in a closed configuration and applying an        external force over at least part of the plates; and    -   analyzing the analysts in the layer of uniform thickness while        the plates are the closed configuration.

In some embodiments, wherein the method comprises:

-   -   (a) obtaining a sample;    -   (b) obtaining a first and second plates that are movable        relative to each other into different configurations, wherein        each plate has a sample contact surface that is substantially        planar, one or both plates are flexible, and one or both of the        plates comprise spacers that are fixed with a respective sample        contacting surface, and wherein the spacers have:        -   i. a predetermined substantially uniform height,        -   ii. a shape of pillar with substantially uniform            cross-section and a flat top surface;        -   iii. a ratio of the width to the height equal or larger than            one;        -   iv. a predetermined constant inter-spacer distance that is            in the range of 10 um to 200 um;        -   v. a filling factor of equal to 1% or larger; and    -   (c) depositing the sample on one or both of the plates when the        plates are configured in an open configuration, wherein the open        configuration is a configuration in which the two plates are        either partially or completely separated apart and the spacing        between the plates is not regulated by the spacers;    -   (d), after (c), using the two plates to compress at least part        of the sample into a layer of substantially uniform thickness        that is confined by the sample contact surfaces of the plates,        wherein the uniform thickness of the layer is regulated by the        spacers and the plates, and has an average value equal to or        less than 30 um with a variation of less than 10%, wherein the        compressing comprises:        -   bringing the two plates together, and        -   conformable pressing, either in parallel or sequentially, an            area of at least one of the plates to press the plates            together to a closed configuration, wherein the conformable            pressing generates a substantially uniform pressure on the            plates over the at least part of the sample, and the            pressing spreads the at least part of the sample laterally            between the sample contact surfaces of the plates, and            wherein the closed configuration is a configuration in which            the spacing between the plates in the layer of uniform            thickness region is regulated by the spacers; and    -   (e) analyzing the in the layer of uniform thickness while the        plates are the closed configuration;    -   wherein the filling factor is the ratio of the spacer contact        area to the total plate area;    -   wherein a conformable pressing is a method that makes the        pressure applied over an area is substantially constant        regardless the shape variation of the outer surfaces of the        plates; and    -   wherein the parallel pressing applies the pressures on the        intended area at the same time, and a sequential pressing        applies the pressure on a part of the intended area and        gradually move to other area.

In some embodiments, wherein the method comprises

-   -   removing the external force after the plates are in the closed        configuration; and    -   imaging the analytes in the layer of uniform thickness while the        plates are the closed configuration; and    -   counting a number of analytes or the labels in an area of the        image.

In some embodiments, wherein the method comprises

-   -   removing the external force after the plates are in the closed        configuration; and    -   measuring optical signal in the layer of uniform thickness while        the plates are the closed configuration.

In some embodiments, the inter-spacer distance is in the range of 20 □mto 200 □m. In some embodiments, the inter-spacer distance is in therange of 5 □m to 20 □n.

In some embodiments, a product of the filling factor and the Young'smodulus of the spacer is 2 MPa or larger.

In some embodiments, the surface variation is less than 50 nm.

In some embodiments, the method further comprising a step of calculatingthe concentration of an analyte in the relevant volume of sample,wherein the calculation is based on the relevant sample volume definedby the predetermined area of the storage site, the uniform samplethickness at the closed configuration, and the amount of target entitydetected.

In some embodiments, the analyzing step comprise counting the analyte inthe sample.

In some embodiments, the imaging and counting is done by:

-   -   i. illuminating the cells in the layer of uniform thickness;    -   ii. taking one or more images of the cells using a CCD or CMOS        sensor    -   iii. identifying cells in the image using a computer; and    -   iv. counting a number of cells in an area of the image.

In some embodiments, the external force is provided by human hand. Insome embodiments, the method future comprises a dry reagent coated onone or both plates.

In some embodiments, the layer of uniform thickness sample has athickness uniformity of up to +/−5%.

In some embodiments, the spacers are pillars with a cross-sectionalshape selected from round, polygonal, circular, square, rectangular,oval, elliptical, or any combination of the same.

In some embodiments, the spacing between the spacers is approximatelythe minimum dimension of an analyte.

The method of any prior claim, wherein one or both plate sample contactsurfaces comprises one or a plurality of amplification sites that areeach capable of amplifying a signal from the analyte or a label of theanalyte when the analyte or label is within 500 nm from an amplificationsite.

In some embodiments, the sample is exhale breath condensate. In someembodiments, the sample is a vapor from a biological sample, anenvironmental sample, a chemical sample, or clinical sample. In someembodiments, the analyte comprises a molecule (e.g., a protein,peptides, DNA, RNA, nucleic acid, or other molecules), cells, tissues,viruses, and nanoparticles with different shapes. In some embodiments,the analyte comprises volatile organic compounds (VOCs). In someembodiments, the analyte comprises nitrogen, oxygen, CO2, H2O, and inertgases. In some embodiments, the analyte is stained.

In some embodiments, on one of the sample surface, it further comprisesan enclosure-spacer that encloses a partial or entire VC samplesdeposited on the collection plate. In some embodiments, the highlyuniform thickness has a value equal to or less than 0.5 um. In someembodiments, the highly uniform thickness has a value in the range of0.5 um to 1 um. In some embodiments, the highly uniform thickness has avalue in the range of 1 um to 2 um. In some embodiments, the highlyuniform thickness has a value in the range of 2 um to 10 um. In someembodiments, the highly uniform thickness has a value in the range of 10um to 20 um. In some embodiments, the highly uniform thickness has avalue in the range of 20 um to 30 um.

In some embodiments, on one of the sample surface, it further comprisesan enclosure-spacer that encloses a partial or entire VC samplesdeposited on the collection plate.

Embodiment (EBC)-1. SiEBCA (Single-Drop Exhaled Breath CondensateCollector and Analyzer)

Exhaled breath condensate (EBC) analysis is a noninvasive method ofdetecting biomarkers, mainly coming from the lower respiratory tract.EBC is collected during quiet breathing, as a product of cooling andcondensation of the exhaled aerosol.

An exemplary method of collecting exhaled breath condensate (EBC) usinga SiEBCA (Single-drop EBC Collector/Analyzer), as illustrated in FIG. 1,comprises the basic steps:

-   -   (1) exhaling breath onto the collection plate (FIG. 1-1). A        subject (e.g. human) breathe onto a plate, termed “collection        plate”, and the breath condenses into EBC, which are in droplets        with different sizes, depending on the breathing time. For a        short breathing time most droplets are separated from each        other. The surface of the collection plate that collects the EBC        is termed the sample surface;    -   (2) placing a cover plate over the collection plate and pressing        them together (FIG. 1-2). A cover plate with spacers (which are        used for regulating the spacing between the cover plate and the        substrate plate) is placed on top of the sample surface; and    -   (3) pressing plates into a “Closed-Configuration (FIG. 1-3). The        cover plate and the substrate are compressed together with at        least a part of the EBC between the plates.

In the method of FIG. 1, the initial droplets are pressed into a thinlayer EBC of a thickness that is regulated by the plates and spacers(not shown).

One reason for using the wording of “single drop” in SiEBCA is that inprinciple, the SiEBCA can detect and analyze a single drop of EBCdeposited on the plate.

In the description of the present invention, “the substrate plate” and“the cover plate” are respectively interchangeable with “the firstplate” and “the second plate”.

In some embodiments, the plates are cooled to reduce the evaporation ofcollected EBC.

A1 A method of collecting EBC, as a basic embodiment of the presentinvention for collecting EBC from a subject, as illustrated in FIG. 1,comprises the steps:

-   -   (a) obtaining a collection plate and a cover plate that are        movable relative to each other into different configurations,        wherein one or both of the plates comprise spacers (not shown in        FIG. 1 but in FIG. 2) that are fixed with the respective plate,        and have a predetermined average height of 100 μm or less;    -   (b) depositing, when the plates are configured in an open        configuration, an EBC sample by exhaling breath from a subject        toward the collection plate, wherein:        -   (i) the exhaled breath condensates on a collection surface            of the collection plate to form droplets and/or puddles that            have different lateral sizes and different heights,            depending upon the surface wetting properties of the            collection surface; and        -   (ii) the open configuration is a configuration in which the            two plates are either partially or completely separated            apart and the spacing between the plates is not regulated by            the spacers; and    -   (c) after (b), bringing the cover plate over the collection        surface of the collection plate and then bringing the two plates        into a closed configuration by pressing the plates, wherein:        -   (i) the closed configuration is a configuration, in which:            at least a part of the spacers are between the cover plate            and the collection plate, and a relevant area of the            collection surface of the collection plate is covered by the            cove plate and has a plate spacing that is regulated by the            spacers; and        -   (ii) at the closed configuration, in the relevant area,            substantial number or all of the droplets or puddles formed            in step (b) at the open configuration mergemerge into            puddle(s) that (1) have much larger lateral size but in a            smaller number than the open configuration and (2) touch            both inner surfaces of the cover plate and the collection            plate, thereby the thickness of the puddle(s) is confined by            the inner surfaces of the plates and equal to the spacing            between the inner surfaces, and the total surface area of            the deposited EBC exposed to the ambient is significantly            reduced;    -   wherein the plate spacing is the spacing between the two inner        surfaces (the two surfaces facing each other) of the cover plate        and the collection plate, the relevant area is a portion or        entire surface of the collection surface, and the collection        surface is a portion or entire surface of the collection plate.

From our experiments (described in details in Examples), we found thatthe final form of the EBC collected by SiEBCA when the plates are in theclosed configuration depends upon the spacer height. Experimentally (seeFIG. 13), we found, as illustrated in FIG. 2, that:

-   -   (1) At the closed configuration of the SiEBCA, if the spacing        between the inner surfaces of the plates is less than the        average height of the EBC droplets or puddles at the open        configuration, the EBC droplets or puddles are compressed by the        collection plate and the cover plate into a continuous film of a        thickness thinner than at the open configuration, and also air        pockets may exist in the film; and    -   (2) otherwise (i.e. if the spacing is equal to or larger than        that the average height at the open configuration) the droplets        and/or puddles self-emerged into discrete puddles that are fewer        in number but larger in lateral size (area) than that in the        open configuration, touch both sample contact (inner) surfaces        of the cover plate and the collection plate, and have the        thickness confined by the inner surfaces of the plates and equal        to the spacing between the inner surfaces. In this case, the EBC        sample thickness at the closed configuration is equal to or        larger than the EBC sample average thickness at the open        configuration. The increase in the EBC puddle thickness at the        closed configuration, as we observed experimentally, are due to        the interactions between the plates and the EBC sample.

EBC-1.2. Device for EBC Collection

FIG. 3. is an illustration of an embodiment of the devices and themethods of a SiEBCA (Single-drop EBC Collector/Analyzer): (a) having afirst plate and a second plate, wherein one or both plate has spacers(shown here: only the first plate has spacers); (b) depositing a sample(only one of many EBC droplets is shown) on the first plate (shown), orthe second plate (not shown), or both (not shown) at an openconfiguration; and (c) (i) using the two plates to spread the sample(the sample flow between the plates) and reduce the sample thickness,and (ii) using the spacers and the plate to regulate the samplethickness at the closed configuration.

A2 A device of collecting EBC, as a basic embodiment of the presentinvention for collecting EBC sample from a subject, as illustrated inFIG. 1, comprises:

-   -   i. a first plate and a second plate, wherein the plates are        movable relative to each other into different configurations,        and one or both plates are flexible;    -   ii. a sample contact area on the respective surface of each of        the plates for contacting EBC sample,    -   iii. spacers on one or both of the plates, wherein the spacers        are fixed with a respective plate, have a predetermined        substantially uniform height of 30 um or less and a        predetermined constant inter-spacer distance that is 250 μm or        less, and wherein at least one of the spacers is inside the        sample contact area;    -   wherein one of the configurations is an open configuration, in        which: the two plates are separated apart, the spacing between        the plates is not regulated by the spacers, and the EBC sample        is deposited on one or both of the plates from a subject; and    -   wherein another of the configurations is a closed configuration        which is configured after the EBC sample deposition in the open        configuration; and in the closed configuration: at least part of        the EBC sample is compressed by the two plates into a layer of        highly uniform thickness, wherein the uniform thickness of the        layer is in contact with and confined by the inner surfaces of        the two plates and is regulated by the plates and the spacers.

In some embodiments of paragraphs A1 and A2, the deposition of the EBCsample is by directly exhaling from a subject to one of the plates.

In some embodiments of paragraphs A1 and A2, the deposition of the EBCsample is by directly exhaling from a subject to both of the plates.

“Covering time delay” means a time period that it takes from the step(b) EBC deposition of paragraph A1 to the end of the step (c) ofparagraph A1 that brings the cover plate and the collection plate to aclosed configuration.

In the method of paragraph A1, the covering time delay should be asshort as possible to reduce the evaporation of deposited EBC. In onepreferred embodiment, the covering time delay is equal to or less than 2sec. In another preferred embodiment, the covering time delay is equalto or less than 5 sec. In another preferred embodiment, the coveringtime delay is equal to or less than 10 sec. In another preferredembodiment, the covering time delay is equal to or less than 30 sec. Andin another preferred embodiment, the covering time delay is in the rangeof 30 sec to 300 sec (e.g. 30 to 60 sec, 60 sec to 120 sec, or 120 secto 300 sec).

EBC-1.3. Significant Reduction of EBC Evaporation Rate

One key advantage of the method and device of paragraph A1 and A2 isthat, compared to the open configuration of the collection plate and thecover plate, the closed configuraiotn of the plates significantlyreduces the surface area of the EBC exposed to the ambient, and hencesignificantly reduces the EBC sample evaporation rate and significantlyincreases the time that EBC sample is in liquid form (i.e. the time thatEBC sample is not completely evaporated). For example, we have observedthat the drying time (the time it takes for the EBC sample to dry outcompletely) increased from 30 secs at an open configuration to 70 mins,a factor of 140 times longer.

EBC-1.4. Guard Ring (Enclosed Spacers)

To further reduce the EBC sample evaporation rate, the enclosed spacersor the guard rings can be used to surround the sample to seal off thesample from the ambient. The guard ring can circle an area that is thesame as, or larger or smaller than the EBC sample deposited at the openconfiguration. The guard ring can be configured to further divide an EBCsample into multiple chambers (FIG. 4.).

FIG. 4. is an illustration of a SiEBCA with both “open spacer” andenclosed spacer, where the open spacer is a post (pillar) while theenclosed spacer is a ring. The enclosed spacer reduces the evaporationof the EBC collected inside the enclosed spacer, since at the closedconfiguration, the enclosed spacer, the cover plate and the collectionplate form an enclosed chamber. If there are only the open spacers butnot an enclosed spacer, at the plate closed configuration, the collectedEBC still evaporates from the edge of the film formed by the EBC,although such evaporation is much slower than that without a coverplate.

In the method and the device of paragraph A1 and A2, the spacers can bean open spacer(s), an enclosed spacer(s), or a combination of thereof.

Details of the devices and methods to keep the EBC thickness uniform aregiven in the other part of the disclosure.

EBC-2. EBC Analysis

Another significant advantage of the present invention is that themethod and the device of paragraph A1-2 can be used for as an EBCanalyzer by itself or by certain modifications. The EBC analyzer analyzeone or a plurality of target analytes in the EBC. The target analytesare further discussed in Section 3, . . . .

The modifications made to the method and device of paragraphs 1-2include, but not limited to, the following, which can used alone(individually) or in combinations:

-   -   (1) Binding Sites. One or both of the plates have one or        plurality of binding site . . . . Each (type) of the regents are        in either in well separated locations (the well separation will        be defined later).    -   (2) Storage sites. One or both of the plates have one or        plurality of binding site . . . . Each (type) of the regents are        in either in well separated locations (the well separation will        be defined later).    -   (3) Amplification site.    -   (4) Multiplexing of analyte detections.

More details of the binding sites, storage sites, amplification sites,and multuplixing sites, as well as their usage for VC and EBC analysisare given in the other part of the disclosure.

A-3 A method of analyzing EBC from a subject for analyzing EBC of asubject comprising:

-   -   (a) obtaining a collection plate and a cover plate that are        movable relative to each other into different configurations,        wherein one or both of the plates comprise spacers (not shown in        FIG. 1 but in FIG. 2) that are fixed with the respective plate,        and have a predetermined average height of 100 μm or less;    -   (b) depositing, when the plates are configured in an open        configuration, an EBC sample by exhaling breath from a subject        toward the collection plate, wherein:        -   (i) the exhaled breath condensates on a collection surface            of the collection plate to form droplets and/or puddles that            have different lateral sizes and different heights,            depending upon the surface wetting properties of the            collection surface; and        -   (ii) the open configuration is a configuration in which the            two plates are either partially or completely separated            apart and the spacing between the plates is not regulated by            the spacers;    -   (c) after (b), bringing the cover plate over the collection        surface of the collection plate and then bringing the two plates        into a closed configuration by pressing the plates, wherein:        -   (i) the closed configuration is a configuration, in which:            at least a part of the spacers are between the cover plate            and the collection plate, and a relevant area of the            collection surface of the collection plate is covered by the            cove plate and has a plate spacing that is regulated by the            spacers; and        -   (ii) at the closed configuration, in the relevant area,            substantial number or all of the droplets or puddles formed            in step (b) at the open configuration merge into puddle(s)            that (1) have much larger lateral size but in a smaller            number than the open configuration and (2) touch both inner            surfaces of the cover plate and the collection plate,            thereby the thickness of the puddle(s) is confined by the            inner surfaces of the plates and equal to the spacing            between the inner surfaces, and the total surface area of            the deposited EBC exposed to the ambient is significantly            reduced; and    -   (d) analyzing the EBC,    -   wherein the plate spacing is the spacing between the two inner        surfaces (the two surfaces face each other) of the cover plate        and the collection plate, the relevant area is a portion or        entire surface of the collection surface, and the collection        surface is a portion or entire surface of the collection plate.

The collection plate generally is held at a temperature the same as theambient, but in some embodiments, the temperatures can be different fromthe ambient, either higher or lower depending upon the goal of thecollection. For example, a temperature lower than the ambient may beused for reducing the EBC evaporation; and a temperature higher than theambient many bused for evaporating more than that at the ambienttemperature is needed.

EBC-3. Applications

EBC analysis can be used for detection of inflammatory markers, whichreflect the state of chronic airways diseases such as chronicobstructive pulmonary disease (COPD), asthma, and cystic fibrosis (CF).EBC analysis can also be used for identification of metabolic,proteomic, and genomic fingerprints of breathing, aiming for an earlydiagnosis of not only respiratory, but also systemic diseases.

EBC-3.1. Analysis of EBC

Breath tests are among the least invasive methods available for clinicaldiagnosis, disease state monitoring, health monitoring and environmentalexposure assessment.

A breath matrix from a subject is a mixture of nitrogen, oxygen, CO2,H2O, and inert gases. The remaining small fraction consists of more than1000 trace volatile organic compounds (VOCs) with concentrations in therange of parts per million (ppm) to parts per trillion (ppt) by volume.In terms of their origin, these volatile substances may be generated inthe body (endogenous) or may be absorbed as contaminants from theenvironment (exogenous). The composition of VOCs in breath varies widelyfrom person to person, both qualitatively and quantitatively.

Although the number of VOCs found to date in human breath is more than1000, only a few VOCs are common to all humans. These common VOCs, whichinclude isoprene, acetone, ethane, and methanol, are products of coremetabolic processes and are very informative for clinical diagnostics.The bulk matrix and trace VOCs in breath exchange between the blood andalveolar air at the blood-gas interface in the lung. One exception isNO, which is released into the airway in the case of airwayinflammation.

The endogenous compounds found in human breath, such as inorganic gases(e.g., NO and CO), VOCs (e.g., isoprene, ethane, pentane, acetone), andother typically nonvolatile substances such as isoprostanes,peroxynitrite, or cytokines, can be measured in breath condensate.Testing for endogenous compounds can provide valuable informationconcerning a possible disease state. Furthermore, exogenous molecules,particularly halogenated organic compounds, can indicate recent exposureto drugs or environmental pollutants.

Volatile Organic Compounds (VOCs) are organic substances that have ahigh vapor pressure and therefore evaporate at room temperature. TheVOCs that may be assayed as target analytes by the methods and devicesprovided by the present invention include, but not limited to,biologically generated VOCs (e.g., terpenes, isoprene, methane, greenleaf volatiles) and anthropogenic VOCs (e.g., typical solvents used inpaints and coatings, like ethyl acetate, glycol ethers, and acetone,vapors from adhesives, paints, adhesive removers, building materials,etc., like methylene chloride, MTBE, and formaldehyde, chlorofurocarbonsand perchloroethylene used in dry cleaning, vapor and exhaustive gasfrom fossil fuels, like benzene and carbon monoxide). Detaileddiscussions on certain biomarkers is given in Table 1.

TABLE 1 Breath markers in certain diseases or applications. Disease orapplication Breath marker Oxidative Lipid peroxidation Pentane, ethanestress Asthma, COPD, bronchiectasis, H₂O₂ breath ARDS methylated alkanecontour Lung Asthma NO, CO, H₂O₂, diseases isoprostanes, nitrite/nitrate COPD NO, H₂O₂, eicosanoids (leukotrienes, prostanoids,isoprostanes), isoprostanes Cystic fibrosis NO, CO, H₂O₂, isoprostanes,nitrite/ nitrate Pulmonary allograft NO dysfunction Lung cancer NO Lungtransplant recipient Exhaled carbonyl sulfide with acute rejectionMetabolic Diabetes Acetone diseases Gastro- Disorders of digestion andH₂ enteric absorption (lactase deficiency, diseases disorders of di- andmono- saccharide malabsorption, starch malabsorption, and small-bowelbacterial overgrowth) Gastritis, duodenal ulcer, gastric Isotopes ofcarbon (¹³C or ulcer, and gastric cancer ¹⁴C) Assess- Vinyl chloride andcis-1,2- ment of dichloroethene, chloroform exposure andbromodichloromethane, to VOCs trichloroethene Other Respiratorymonitoring CO₂/O₂ ratio applica- Excretion of drugs tions

EBC-3.2. Collection and Analysis of Other Vapor Condensates

Certain embodiments of the present invention are related to theapplications of the SiEBCA methods and devices for collection andanalysis of the vapor condensates other than the EBC. The othermoistures include, but not limited to, fog, clouds, steams, etc. Thetarget analysis of these vapor condensates can be for different purposeenvironmental monitoring, emission control, etc. In some embodiments,the sample is a vapor from a biological sample, an environmental sample,a chemical sample, or clinical sample.

EBC-3.3. Automatic and High Throughput

In certain embodiments, the devices and methods of the present inventionare automatic and high speed, where the steps are performed by machines.In some embodiments, the plates are in the form of roll of sheets andare controlled by rollers to put certain area of the plates into an openconfiguration or a closed configuration.

EBC-4. More Examples of EBC Collection and Analysis Experiments

Additional exemplary experimental testing and observation, andadditional preferred embodiments of the present invention are given.

All the exemplary experimental testing and demonstration of the presentinvention described in Section 4 (Examples) were performed under thefollowing conditions and share the following common observations.

Plates.

Only one of the two plates of SiEBCA device, termed “X-Plate”, has thespacers fixed on the sample surface of the plate, and the other plate,termed “the substrate plate”, has a planar surface and does not havespacers. The substrate plate was used as the collection plate, and theX-plate was used as the cover plate. Various materials (including glass,PMMA (polymethacrylate), and PS (polystyrene)) for the plates andvarious plate thicknesses have been tested. The planar surface of theplates typically have surface roughness less than 30 nm.

Spacers.

The spacers used on the X-Plate are rectangle pillars in a periodicarray with a fixed inner spacer distance (ISD) and uniform spacerheight. The pillar spacers have a straight sidewall with a tilt anglefrom the normal less than 5 degree. Different spacer height, size,inter-spacer distance, shape, and materials are tested.

Fabrication of Spacers.

The spacers are fabricated by nanoimprint on a plastic plate, where amold is pressed directly into the plate. The mold was fabricated bylithography and etching. Examples of the spacers on the plate forSiEBCA. The spacers are fabricated by direct imprinting of the plasticplate surface using a mold, and has a dimension of width, length andheight of 30 um, 40 um and 2 um.

EBC Sample Deposition.

All of the EBC samples were deposited on the collection plates by havinga human subject directly exhale toward the collection plate which isplaced within a few inches away from the subject's mouth.

The EBC samples depositions were performed in standard room conditionswithout any special temperature control or dust filters. We found thatin our experiments, the dust does not affect to achieve thepredetermined final sample thickness over a large sample area, and atthe closed configuration the sample thickness over the non-dust area isregulated by the spacers. This demonstrated that the embodiments that weused for CROF performed to the results expected by the presentinvention.

Plate's Surface Wetting Properties.

Unless particularly specified, all the sample surfaces (i.e. the innersurface that contacts a sample) of the plates are untreated. We havetested the wetting properties of these untreated surfaces as a functionof the plate material (glass, PMMA, and PS), the surface structures(planar or with spacers, and the sample type (water, PBS buffer andblood), by dropping a small drop of sample on the plate surface andmeasuring the sample to the plate contact angle. The wetting angles ofthe different surfaces for different samples were found experimentallyas follows: For the liquid of water, PBS, and blood, the contact angleis about 46 degree for untreated glass, 60 degree for untreated PMMAsurface, 60 degree for untreated PS (polystyrene) and about 61 degreesfor untreated PMMA X-plate. Therefore they are all hydrophilic. But thewetting property of these surfaces can changed to either hydrophilic orhydrophobic by surface treatment. For a good vapor condensatecollection, a hydrophilic surface is preferred, which will have, for agiven amount of the condensation, smaller surface area tha thHand-Press.In all the experiments in the Section, the plates in a SiEBCA processwere brought together and compressed into a closed configuration of theplates all by human hand(s). In a final pressing for uniform samplethickness over a large area of the SiEBCA plates, often a thumb pressesone area and rubs into different areas of the CROF plates, and excellentsample thickness uniformity were observed as detailed below. A processthat uses hand(s) to press a SiEBCA device (plates) into a closedconfiguration is referred as “hand-pressing”.

FIG. 5 gives examplary methods of pressing the plates of SiEBCA by humanhand. The SiEBCA can be pressed by either (a) placing the SiEBCA on asurface and uses a thumb to press one location of the SiEBCA and pressand rub into other locations, or (b) placing the SiEBCA between a thumband a figure and press and rub. In some cases, both hands can be used.

Self-Holding.

Self-holding means that after a SiEBCA device (plates) is compressedinto a closed configuration by an external force (e.g. the force fromhand) and after the external force is removed, the SiEBCA device canhold, on its own, the sample thickness unchanged. We observed that inall the experiments in the Sec., all the SiEBCA devices and process(unless particularly specified) can self-hold, as demonstrated in theexperiments. Our other experimental test showed that as long as one ofthe plate is hydrophilic, the SiEBCA plates can self-hold.

EBC-4.1. EBC Formation on Collection Plate at Open and ClosedConfigurations 4.1.1 EBC Droplet Size on Untreated and Surface TreatedPMMA Collection Plate

FIG. 6. Experimental data of EBC Droplets sizes and density on thecollection plate (untreated PMMA film) at an “open configuration” (i.e.only the collection plate without the cover plate). The photographs showthe EBC collected on the collection plate after a subject directlybreathe to the plate for four different breathing time: (a) 1 secbreath, (b) 2 sec breath, (c) 5 sec breath, and (d) 10 sec breath. Thephotographs were taken immediately after the breathing.

The table shows the measured average droplet size, the calculatedaverage droplet height, the average drop volume, the measured dropletdensity, and the total liquid surface area on 1 mm-square area of thecollection plate (PMMA). The experimental data show that (1) using auntreated PMMA film as the collection plate's collection surface, theEBC directly from a subject form droplets that have different sizes andare, for most droplets, well separated from each other; and (2) themeasured average droplet size, the calculated average droplet height,the average droplet volume, the droplet density, and the total liquidsurface area on 1 mm-square area of the collection plate (PMMA)initially increase with the breathing time, but seem to become saturatedafter 5 s breathing. This might be due to the fact that in theexperiment, after 5 sec breathing, the EBC deposition rate by breathingand the evaporation rate of the existing EBC reach an equilibrium.

In the above experiment, the calculation of the average droplet heightis based on the wetting contact angle of water on PMMA, and the volumedensity is calculated by multiplying the average droplet volume with themeasured droplet amount density.

The table also shows that the average EBC volume is 172 pL, 250 pL, 491pL, and 507 pL per sq-mm collection plate area, respectively, for 1 s, 2s 5 s and 10 s breathing time.

FIG. 7. Experimental data of EBC formation on the collection plate,which is a surface treated PMMA film (the treatment made the surfacemore hydrophilic than an untreated PMMA film surface), at a plate openconfiguration. The photographs show that the EBC collected on thecollection plate from a direct breathing by a subject for four differentbreathing time: (a) 1 sec breath, (b) 2 sec breath, (c) 5 sec breath,and (d) 10 sec breath. The photographs were taken immediately after thebreathing. Assuming there were the same amount of EBC deposited on thecollection plate with treated surface as in the case of untreatedsurface, the average liquid thickness was therefore calculated bydividing the total volume by the observed liquid sample area on the 1mm² collection plate. The table shows the volume density per unitsurface area, the calculated average liquid thickness, and the totalliquid surface area on 1 mm² area of the collection plate (PMMA).

The experimental data in FIG. 7 clearly show that: (1) due to thehydrophilic surface with smaller contact angle of the treated PMMA(details given in FIG. 8), the EBC deposited directly from a subject tothe collection plate forms, at an open configuration, a few large-areaEBC puddles rather than many small droplets; and (2) the total liquid(EBC) surface area on 1 mm-square area for the EBC collected using thecollection plate with surface treatment (hence bettering wetting) isabout 4 times less that without the surface treatment.

FIG. 8. Photographs and measured evaporation time (at plate openconfiguration) of the EBC (2 s breathing directly from a subject)collected on untreated and treated PMMA plate. The photographs show thatthe EBC collected on the untreated PMMA collection plate form many smallwell separated droplets (a), while the EBC collected on the treated PMMAcollection plate form a few thin film and large puddles with large voids(b). The calculated surface area of the EBC collected on the untreatedsurface is 4 times larger than that on the untreated surface. And theEBC collected on the untreated surface has a total evaporation time of 7s, which is about 4 times shorter than that collected on the treatedsurface, which is 30 s. More studies of the evaporation time are givenin FIGS. 13 and 14 and described below.

4.1.2 EBC Formation when the Plates are in a Closed Configuration

Experimentally, we observed that the final form of the EBC collected bySiEBCA when the plates are in the closed configuration depends upon thespacer height.

FIGS. 9 and 10 show, respectively, the photographs and experimental dataof spacer height effects (1 um, 2 um, 10 um and 30 um, respectively) onthe EBC collected using SiEBCA at the closed configuration. Thebreathing time is 2 s and the covering time delay is nearly 0 s. Thespacers are pillars of uniform cross-section (30 um×38 um) with flat topand bottom surfaces and a constant inter spacer distance of 80 um and 82um (X and Y direction). The cover plate is an untreated X-plate of 175um thick PMMA film and the collection plate is a flat glass plate (25mm×25 mm×1 mm). As calculated, the average height of the droplets at theopen configuration that are collected on the untreated surface from 2 sbreathing is 1.7 um. Here, the experiments show that:

-   -   (1) For all the spacer heights tested (shown in FIG. 9), the EBC        on the collection plate merged from the droplets formed when the        plates were at an open configuration to puddles that are fewer        in number but much larger in lateral size when the plates were        at the closed configuration.    -   (2) 1 um gapping X-device collected the largest amount of breath        liquid with the best liquid thickness uniformity and in        continuous films.    -   (3) X-devices with gapping larger than 1 um collect collected        less breath liquid and were hard to self-hold well, thus had        worse liquid thickness uniformity and deviation from pillar        height.        Therefore, experimentally, we demonstrated that:    -   (1) At the closed configuration of the SiEBCA, if the spacing        between the plate sample surfaces of the plates is less than the        average height of the EBC droplets or puddles at the open        configuration (e.g. the spacing was 1 um and the average height        was 1.7 um), the EBC droplets or puddles are compressed by the        collection plate and the cover plate into a continuous film of a        thickness thinner than the open configuration, and also air        pockets may exist in the film; and    -   (2) otherwise (e.g., the pillar height was 2 um, 10 um, or 30        um, respectively, but the EBC at the open configuraiotn was only        1.7 um height) the droplets and or puddles first raised up to        touch both sample contact (inner) surfaces of the cover plate        and the collection plate, and then self-emerged to discrete        puddles that are fewer in number but larger in lateral size        (area) than that in the open configuration, and have the        thickness confined by the inner surfaces of the plates and equal        to the spacing between the inner surfaces. In this case, the EBC        sample thickness at the closed configuration is equal to or        larger than the EBC sample average thickness at the open        configuration. The increase in the EBC puddle thickness at the        closed configuration, as we observed experimentally, are due to        the interactions between the plates and the EBC sample.

4.1.3 EBC Evaporation Time as Function of the Covering Delay Time

FIGS. 11 and 12 show, respectively, photographs and experimental data ofthe effects on breath collection of (a) treated versus untreated PMMAcollection plates and (b) time delay in closing the cover plate. In theexperiments, the EBC was collected directly from 2 s breathing of asubject on a collection plate (PMMA 25 mm×25 mm×1 mm); some collectionplates were untreated, but some were treated to have a betterhydrophilicity than the untreated PMMA collection plate; the cover platewas an X-plate of 175 um thick PMMA with a square lattice ofpillar-shaped spacers of flat top (1 um height, 30 um width, and 38 umlong) with inter-spacer distance of 80 um and 82 um in x and y directionrespectively; two different time delays in covering the collection platewith the cover plate were tested: immediately after the completion ofthe breath or 5 s after the completion of the breath; and the liquidthickness, the collected breath liquid volume, the Liquid thicknessdeviation from pillar height, Liquid thickness uniformity, and otherparameters (e.g. Liquid Area (mm2) on 25 mm×25 mm collection plate,(Liquid Area) over (Total Area−Spacer Area) were measured all at theplate closed configuration.

Through the experimental study, we found that:

(1) Compared with untreated PMMA collection plate, the treatedcollection plates (better wetting) collect more breath liquid at bothdelay times of “immediate press” and “at 5 s press”.

(2) Using the untreated collection plates (less hydrophilic), at 5 sdelay time for covering, almost a half of the liquid evaporated.

(3) For all the samples, after the hand pressing, the plates in SiEBCAcan self-hold.

(4) In most cases, at the closed configuration, for most of the delaytimes, the measured deviation of the EBC average thickness from thespacer height is equal to or less than 7.4% and the measured EBCthickness uniformity (i.e. variation) is equal to or less than 6.4%. Butwhen the collection surface is untreated (less hydrophilic) and the timedelay for coving the cover plate is 5 s, the average EBC thicknessdeviates from the spacer height is large (1.52 um compared to 1 um,leading to a 52% relative deviation) and the thickness uniformity ispoor 22% variation.

FIG. 13. Experimental data of the volume of the collected breath (i.e.EBC) on the collection plate vs. the time delay (measured from the endof the breath to the covering of the cover plate) in the case of thetreated (which is more hydrophilic that the untreated) and untreatedcollection plate (PMMA) surface, respectively. In the experiments, thebreathing time was 2 s. The collected breath volume was measured afterthe cover plate and the collection plate were pressed to the closedconfiguration. From the experiments, we found that:

(1) For a given time delay, the collection plate with a treated surface(more wetting than untreated) had more breath liquid collected.

(2) Without covering the collection plate, the EBC from the 2 s breathlasted only 7 s for the collection plate with untreated surface, but 30s for the collection plate with treated surface, which is 4 timeslonger.

The observation (2) further shows that with a more wetting surface onthe collection plate, the total surface area of the EBC deposited on theplate has a smaller surface area than that deposited on the untreatedarea, and hence there is a longer drying time on the treated surface(i.e. the time before completely drying out).

4.1.4 EBC Evaporation Time at Plate Open and Closed Configuration

We experimentally studied the evaporation time of the EBC deposited onthe collection plate without and with the cover plate placed on top ofthe EBC (i.e. the evaporation time for the plates being in the openconfiguration and the closed configuration.

The evaporation rate of the EBC on the collection plate at the openconfiguration has been given and described in FIG. 8.

FIG. 14. Experiment Data for drying time of EBC collected by SiEBCA at“Closed configuration” in the case of the treated (which is morehydrophilic that the untreated) and untreated collection plate (PMMA)surface, respectively, and at different spacer height. In theexperiments, the breath time was 2 s., and the cover plate wasimmediately covered dafter the breath. The experiments showed that: thetreated PMMA collection plate with a 1 um spacer allowed a drying timeof 70 s, but the drying time reduced to 45 s if the spacer was 10 um.The reason for a shorter drying time at 10 um spacer is due to the factthat the EBC at the plate closed configuration form many isolatedpuddles, which has more surface area to be exposed to the environmentthan that in a ium spacer height SiEBCA which has one or a few large EBCareas.

We realized that by using an enclosed spacer in addition to the isolatedspacers, we can further reduce the EBC evaporation rate at a closedconfiguration of SiEBCA.

Drying speed with treated and untreated collection plates. For bothexperiments using treated and untreated collection plates, the dryingspeed of EBC was also calculated, which is defined as the retractionlength per unit time of the edge of the liquid sample in the X-device atthe closed configuration. The calculation shows that with the treatedPMMA collection plate, the liquid sample dried at a slower speed (74um/min) as compared to with the untreated PMMA collection plate (117um/min).

Surface treatment. The surface treatment of the PMMA plate was performedwith oxygen plasma, followed by deposition of 10 nm silicon oxide. Insome embodiments, the treatment was performed chemically usingtrimethoxysilane.

Compressed Regulated Open Flow” (CROF)

Many embodiments of the present invention manipulate the geometric size,location, contact areas, and mixing of a sample and/or a reagent using amethod, termed “compressed regulated open flow (CROF)”, and a devicethat performs CROF.

The term “compressed open flow (COF)” refers to a method that changesthe shape of a flowable sample deposited on a plate by (i) placing otherplate on top of at least a part of the sample and (ii) then compressingthe sample between two plates by pushing the two plates towards eachother; wherein the compression reduces a thickness of at least a part ofthe sample and makes the sample flow into open spaces between theplates.

The term “compressed regulated open flow” or “CROF” (or “self-calibratedcompressed open flow” or “SCOF” or “SCCOF”) refers to a particular typeof COF, wherein the final thickness of a part or entire sample after thecompression is “regulated” by spacers, wherein the spacers, that areplaced between the two plates.

The term “the final thickness of a part or entire sample is regulated byspacers” in a CROF means that during a CROF, once a specific samplethickness is reached, the relative movement of the two plates and hencethe change of sample thickness stop, wherein the specific thickness isdetermined by the spacer.

One embodiment of the method of CROF, as illustrated in FIG. 1-4,comprises:

-   -   (a) obtaining a sample, that is flowable;    -   (b) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        each plate has a sample contact surface that is substantially        planar, wherein one or both of the plates comprise spacers and        the spacers have a predetermined height, and the spacers are on        a respective sample contacting surface;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;        and    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers, wherein the relevant volume is at least a portion        of an entire volume of the sample, and wherein during the sample        spreading, the sample flows laterally between the two plates.

The term “plate” refers to, unless being specified otherwise, the plateused in a CROF process, which a solid that has a surface that can beused, together with another plate, to compress a sample placed betweenthe two plate to reduce a thickness of the sample.

The term “the plates” or “the pair of the plates” refers to the twoplates in a CROF process.

The term “first plate” or “second plate” refers to the plate use in aCROF process.

The term “the plates are facing each other” refers to the cases where apair of plates are at least partially facing each other.

The term “spacers” or “stoppers” refers to, unless stated otherwise, themechanical objects that set, when being placed between two plates, alimit on the minimum spacing between the two plates that can be reachedwhen compressing the two plates together. Namely, in the compressing,the spacers will stop the relative movement of the two plates to preventthe plate spacing becoming less than a preset (i.e. predetermined)value. There are two types of the spacers: “open-spacers” and“enclosed-spacers”.

The term “open-spacer” means the spacer have a shape that allows aliquid to flow around the entire perimeter of the spacer and flow passthe spacer. For example, a pillar is an open spacer.

The term of “enclosed spacer” means the spacer of having a shape that aliquid cannot flow abound the entire perimeter of the spacer and cannotflow pass the spacer. For example, a ring shape spacer is an enclosedspacer for a liquid inside the ring, where the liquid inside the ringspacer remains inside the ring and cannot go to outside (outsideperimeter).

The term “a spacer has a predetermined height” and “spacers havepredetermined inter-spacer distance” means, respectively, that the valueof the spacer height and the inter spacer distance is known prior to aCROF process. It is not predetermined, if the value of the spacer heightand the inter-spacer distance is not known prior to a CROF process. Forexample, in the case that beads are sprayed on a plate as spacers, wherebeads are landed on random locations of the plate, the inter-spacerdistance is not predetermined. Another example of not predeterminedinter spacer distance is that the spacers moves during a CROF processes.

The term “a spacer is fixed on its respective plate” in a CROF processmeans that the spacer is attached to a location of a plate and theattachment to that location is maintained during a CROF (i.e. thelocation of the spacer on respective plate does not change). An exampleof “a spacer is fixed with its respective plate” is that a spacer ismonolithically made of one piece of material of the plate, and thelocation of the spacer relative to the plate surface does not changeduring CROF. An example of “a spacer is not fixed with its respectiveplate” is that a spacer is glued to a plate by an adhesive, but during ause of the plate, during CROF, the adhesive cannot hold the spacer atits original location on the plate surface and the spacer moves awayfrom its original location on the plate surface.

The term “a spacer is fixed to a plate monolithically” means the spacerand the plate behavior like a single piece of an object where, during ause, the spacer does not move or separated from its original location onthe plate.

The term “open configuration” of the two plates in a CROF process meansa configuration in which the two plates are either partially orcompletely separated apart and the spacing between the plates is notregulated by the spacers

The term “closed configuration” of the two plates in a CROF processmeans a configuration in which the plates are facing each other, thespacers and a relevant volume of the sample are between the plates, thethickness of the relevant volume of the sample is regulated by theplates and the spacers, wherein the relevant volume is at least aportion of an entire volume of the sample.

The term “a sample thickness is regulated by the plate and the spacers”in a CROF process means that for a give condition of the plates, thesample, the spacer, and the plate compressing method, the thickness ofat least a port of the sample at the closed configuration of the platescan be predetermined from the properties of the spacers and the plate.

The term “inner surface” or “sample surface” of a plate in a CROF devicerefers to the surface of the plate that touches the sample, while theother surface (that does not touch the sample) of the plate is termed“outer surface”.

The term “X-Plate” of a CROF device refers to a plate that comprisesspaces that are on the sample surface of the plate, wherein the spacershave a predetermined inter-spacer distance and spacer height, andwherein at least one of the spacers is inside the sample contact area.

The term “CROF device” refers to a device that performs a CROF process.The term “CROFed” means that a CROF process is used. For example, theterm “a sample was CROFed” means that the sample was put inside a CROFdevice, a CROF process was performed, and the sample was hold, unlessstated otherwise, at a final configuration of the CROF.

The term “CROF plates” refers to the two plates used in performing aCROF process.

The term “surface smoothness” or “surface smoothness variation” of aplanar surface refers to the average deviation of a planar surface froma perfect flat plane over a short distance that is about or smaller thana few micrometers. The surface smoothness is different from the surfaceflatness variation. A planar surface can have a good surface flatness,but poor surface smoothness.

The term “surface flatness” or “surface flatness variation” of a planarsurface refers to the average deviation of a planar surface from aperfect flat plane over a long distance that is about or larger than 10um. The surface flatness variation is different from the surfacesmoothness. A planar surface can have a good surface smoothness, butpoor surface flatness (i.e. large surface flatness variation).

The term “relative surface flatness” of a plate or a sample is the ratioof the plate surface flatness variation to the final sample thickness.

The term “final sample thickness” in a CROF process refers to, unlessspecified otherwise, the thickness of the sample at the closedconfiguration of the plates in a CORF process.

The term “compression method” in CROF refers to a method that brings twoplates from an open configuration to a closed configuration.

The term of “interested area” or “area of interest” of a plate refers tothe area of the plate that is relevant to the function that the platesperform.

The term “at most” means “equal to or less than”. For example, a spacerheight is at most 1 um, it means that the spacer height is equal to orless than 1 um.

The term “sample area” means the area of the sample in the directionapproximately parallel to the space between the plates and perpendicularto the sample thickness.

The term “sample thickness” refers to the sample dimension in thedirection normal to the surface of the plates that face each other(e.g., the direction of the spacing between the plates).

The term “plate-spacing” refers to the distance between the innersurfaces of the two plates.

The term “deviation of the final sample thickness” in a CROF means thedifference between the predetermined spacer height (determined fromfabrication of the spacer) and the average of the final samplethickness, wherein the average final sample thickness is averaged over agiven area (e.g. an average of 25 different points (4 mm apart) over 1.6cm by 1.6 cm area).

The term “uniformity of the measured final sample thickness” in a CROFprocess means the standard deviation of the measured final samplethickness over a given sample area (e.g. the standard deviation relativeto the average.).

The term “relevant volume of a sample” and “relevant area of a sample”in a CROF process refers to, respectively, the volume and the area of aportion or entire volume of the sample deposited on the plates during aCROF process, that is relevant to a function to be performed by arespective method or device, wherein the function includes, but notlimited to, reduction in binding time of analyte or entity, detection ofanalytes, quantify of a volume, quantify of a concentration, mixing ofreagents, or control of a concentration (analytes, entity or reagents).

The term “some embodiments”, “in some embodiments” “in the presentinvention, in some embodiments”, “embodiment”, “one embodiment”,“another embodiment”, “certain embodiments”, “many embodiments”, oralike refers, unless specifically stated otherwise, to an embodiment(s)that is (are) applied to the entire disclosure (i.e. the entireinvention).

The term “height” or “thickness” of an object in a CROF process refersto, unless specifically stated, the dimension of the object that is inthe direction normal to a surface of the plate. For example, spacerheight is the dimension of the spacer in the direction normal to asurface of the plate, and the spacer height and the spacer thicknessmeans the same thing.

The term “area” of an object in a CROF process refers to, unlessspecifically stated, the area of the object that is parallel to asurface of the plate. For example, spacer area is the area of the spacerthat is parallel to a surface of the plate.

The term “lateral” or “laterally” in a CROF process refers to, unlessspecifically stated, the direction that is parallel to a surface of theplate.

The term “width” of a spacer in a CROF process refers to, unlessspecifically stated, a lateral dimension of the spacer.

The term “a spacer inside a sample” means that the spacer is surroundedby the sample (e.g. a pillar spacer inside a sample).

The term “critical bending span” of a plate in a CROF process refers thespan (i.e. distance) of the plate between two supports, at which thebending of the plate, for a given flexible plate, sample, andcompression force, is equal to an allowed bending. For example, if anallowed bending is 50 nm and the critical bending span is 40 um for agiven flexible plate, sample, and compression force, the bending of theplate between two neighboring spacers 40 um apart will be 50 nm, and thebending will be less than 50 nm if the two neighboring spacers is lessthan 40 um.

The term “flowable” for a sample means that when the thickness of thesample is reduced, the lateral dimension increases. For an example, astool sample is regarded flowable.

In some embodiments of the present invention, a sample under a CROFprocess do not to be flowable to benefit from the process, as long asthe sample thickness can be reduced under a CROF process. For anexample, to stain a tissue by put a dye on a surface of the CROF plate,a CROF process can reduce the tissue thickness and hence speed up thesaturation incubation time for staining by the dye.

1. Reducing (Shortening) Binding or Mixing Time (X)

It is desirable to reduce the incubation/reaction time in performingassays or other chemical reactions. For example, in the surfaceimmobilization assays where a target analyte in a sample is detected bybeing captured by capture agents immobilized on a plate surface (i.e. asolid phase), it is often desirable to have a short saturationincubation time for capturing target analytes in the sample, orimmobilizing of the capture agents and detection agents in a solution ona plate surface, or both. Another example is the need to shorten thetime of coating a capture agent to a plate surface. And another exampleis the need to shorten the time of mixing a reagent into a sample.

The present invention provides the methods and devise that reduce (i.e.shorten) the saturation incubation time needed for binding an entity insample to a binding site on a solid surface (i.e. the time for an entityfrom a volume to a surface). Another aspect of the present invention isto reduce the time needed for a binding of an entity stored on a platesurface to a binding site on another plate surface (i.e. the time for anentity from one surface to another surface). Another aspect of thepresent invention is to reduce the time needed for adding/mixing of areagent stored on a surface into a volume of a sample (i.e. a time foradding/mixing a reagent from a surface into a volume of a sample).

The present invention reduces the saturation incubation time of bindingand/or mixing in an assay by using the devices and methods that spread asample (or a liquid) to a thinner thickness, thereby reducing the timefor an entity diffusing across the sample's thickness. A diffusion timeof an entity in a material (e.g. liquid or solid or semi-solid) isproportional to the square to the diffusion distance, hence a reductionof the sample thickness can reduce the diffusion distance, leading todrastically reduction of diffusion time and the saturation incubationtime. A thinner thickness (e.g. a tight confined space) also increasesthe frequency of collisions of an entity with other entities in amaterial, further enhancing a binding and a mixing. Themeans in thepresent invention also make the reduction of the sample's thicknessprecise, uniform, fast, simple (less operation steps) and applicable toreduce the sample thickness to micrometer or nanometer thick. Theinventions have great utilities in fast, low-cost, PoC, diagnostics andchemical/bio analysis. Several embodiments of the present invention areillustrated in FIG. 1-4.

1.1 Reducing the Saturation Incubation Time of Binding an Entity in aSample to a Binding Site on a Solid Surface by Reducing the SampleThickness.

X1. A method for reducing the saturation incubation time of binding atarget entity in a sample to a binding site of a plate surface, asillustrated in FIGS. 1-4 and 15, comprising:

-   -   (a) obtaining a sample that is flowable and contains a target        entity which is capable of diffusing in the sample;    -   (b) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a binding site that is        configured to bind the target entity, wherein one or both of the        plates comprise spacers, and each of the spacers is fixed with        its respective plate and has a predetermined height;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the binding site is        in contact with the relevant volume, and the thickness of the        relevant volume of the sample is regulated by the plates and the        spacers, is thinner than the maximum thickness of the sample        when the plates are in the open configuration;        -   wherein the relevant volume is a portion or an entire volume            of the sample; and        -   wherein the reduced thickness of the sample reduces the            saturation incubation time for binding of the target entity            in the relevant volume to the binding site.

For a given sample volume, a CROF reduces sample thickness but increasethe sample lateral dimension. The present invention utilize the fact toperform (a) local binding or mixing in portion of the sample, and (b)multiplexing of multiple binding or mixing sites, without a fluidicbarrier to fluidically separate a sample into different isolation liquidpockets.

X2. A device for reducing the saturation incubation time to bind targetentity in a relevant volume of a sample to a surface, as illustrated inFIGS. 1-4 and 15, comprising:

-   -   a first plate and a second plate that (a) are movable relative        to each other into different configurations, (b) each plate has        a sample contact area for contacting a sample that has a target        entity in a relevant volume of the sample, (c) one of the plate        has binding site that binds the target entity, and (d) at least        one of the plates comprises spacers that have a predetermined        inter-spacer distance and height and are fixed on its respective        surface, wherein at least one of the spacers is inside the        sample contact area;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, and the spacing between the plates is not        regulated by the spacers,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in an open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the binding site is in contact        with the relevant volume, and the thickness of the relevant        volume of the sample is regulated by the plates and the spacers,        is thinner than the maximum thickness of the sample when the        plates are in the open configuration; wherein the relevant        volume is a portion or an entire volume of the sample; and        wherein the reduced thickness of the sample reduces the        saturation incubation time for a binding of the target entity in        the relevant volume to the binding site.

1.2 Reducing Saturation Incubation Time for a Binding of an EntityStored on One Plate Surface to a Binding Site on Another Plate Surface

X3. A method for reducing the saturation incubation time to bind anentity stored on a storage site of one plate to a relevant binding siteon another plate, as illustrated in FIGS. 1-4 and 15 b, comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein a        surface of first plate has a binding site, and a surface of the        second plate has a storage site that contains an entity to be        bound to the binding site; wherein the area of the binding site        and the area of the storage site is less than that of respective        plates; and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   (b) obtaining a transfer medium, wherein the entity on the        storage site are capable of being dissolving into the transfer        medium and diffusing in the transfer medium;    -   (c) depositing, when the plates are configured in an open        configuration, the transfer medium on one or both of the plates;        wherein the open configuration is a configuration in which the        two plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the transfer medium by bringing the        plates into a closed configuration, wherein, in the closed        configuration: the plates are facing each other, the spacers,        the binding site, the storage site and at least a portion of the        transfer medium are between the plates, the binding site and the        storage site are at least partially on top of each other, the        transfer medium contacts at least a part of the binding site and        the storage site, the thickness of the transfer medium is        regulated by the plates and the spacers, is thinner than the        maximum thickness of the transfer medium when the plates are in        the open configuration;        -   wherein the reduced thickness of the transfer medium reduces            the time for the binging of the entity stored on the second            plate to the binding site on the first plate.

X4. A device for reducing the saturation incubation time for binding anentity stored on a storage site of one plate to a binding site onanother plate, as illustrated in FIGS. 1-4, and 15 b, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations, wherein a surface of        first plate has a binding site; and a surface of the second        plate has a storage site that contains an entity to be bound to        the binding site; wherein the area of the binding site and the        area of the storage site is less than that of respective plates;        and wherein one or both of the plates comprise spacers and each        of the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and a transfer medium can be deposited on one or        both of the plates, wherein the entity on the storage site are        capable of being dissolving into the transfer medium and        diffusing in the transfer medium,    -   wherein another of the configuration is a closed configuration,        which is configured after the transfer medium deposition in an        open configuration; and in the closed configuration: the plates        are facing each other, the spacers, the binding site, the        storage site and at least a portion of the transfer medium are        between the plates, the binding site and the storage site are at        least partially on top of each other, the transfer medium        contacts at least a part of the binding site and the storage        site, the thickness of the transfer medium is regulated by the        plates and the spacers, is thinner than the maximum thickness of        the transfer medium when the plates are in the open        configuration;    -   wherein the reduced thickness of the transfer medium reduces the        saturation incubation time for a binging of entity on the        storage site of the second plate to the binding site of the        first plate.

In the method of paragraph X3 and the device of paragraph X4, in someembodiments, the transfer medium comprises a liquid that allows adiffusion of the entity or a reagent or both.

In the method of paragraph X3 and the device of paragraph X4, in someembodiments, the transfer medium is a sample, where the sample containsan analyte (also termed target analyte) that binds the binding site.

In the method of paragraph X3 and the device of paragraph X4, in someembodiments, the transfer medium is a sample, where the sample containsan analyte (also termed target analyte) that binds the binding site andthe reagent is a detection agent that binds to the analytes.

1.3 Reducing the Time for Adding (Mixing) Reagent Stored on Surface intoa Liquid Sample

Many assays need to have reagents added into a sample (including aliquid). Often the concentration of the added reagents in the sample orthe liquid need to be controlled. There are needs for new methods thatare simple and/or low cost to perform such reagents addition andconcentration control. Two examples where reagents additions are neededare (a) blood cell counting where anticoagulant and/or stainingreagent(s) may be added into a blood sample, and (b) immunoassays wheredetection agents are added to bind a target analyte in solution.

One aspect of the present invention is the methods, devices, and systemsthat make the reagent addition and the reagent concentration controlsimple and/or low cost. In one embodiment of the current invention, areagent layer (e.g. dried reagent layer) is first put on a plate surfaceof a CROF device, then a sample is deposited into the CROF device, and aCROF process makes the sample in contact with the reagent and the samplethickness thinner than the thickness when the sample at the openconfiguration of the CROF plates. By reducing the sample thickness, itwould reduce the diffusion time of the reagent diffuses from the surfaceinto the entire sample, and hence it reduces the time for mixing thereagent with the sample.

X5. A method for reducing the time for mixing a reagent stored on aplate surface into a sample, as illustrated in FIGS. 1-4, and 15 c,comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a storage site that        contains reagents to be added into a sample, and the reagents        are capable of being dissolving into the sample and diffusing in        the sample; and wherein one or both of the plates comprise        spacers and each of the spacers is fixed with its respective        plate and has a predetermined height;    -   (b) obtaining the sample;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers, the storage site,        and at least a portion of the sample are between the plates, the        sample contacts at least a portion of the storage site, the        thickness of the sample on the storage site is regulated by the        plates and the spacers, is thinner than the maximum thickness of        the sample when the plates are in the open configuration;    -   wherein the reduced thickness of the sample reduces the time for        mixing the reagents on the storage site with the sample.

In the method of paragraph X5, it further comprises a step of incubationwhile the plates are in the closed configuration, wherein the incubationtime is selected in such that results in a significant number of thereagents dissolved in the sample are contained in the relevant volume ofthe sample, wherein the relevant volume is the volume of the sample thatsits on the storage site and the incubation is a process to allow thereagent to dissolve and diffuse in the sample.

In the method of paragraph X5, it further comprises a step that, after(d) and while the plates are in the closed configuration, incubating fora time equal or less than a factor times the diffusion time of thereagent in the sample across the sample thickness regulated by theplates at the closed configuration, and then stopping the incubation;wherein the incubation allows the reagent to diffuse into the sample;and wherein the factor is 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3,1.5, 2, 3, 4, 5, 10, 100, 1000, 10,000, or a range between any to thevalues. For example, if the factor is 1.1 and the diffusion time is 20seconds, then the incubation time is equal to or less than 22 second. Inone preferred embodiment, the factor is 0.1, 1, 1.5 or a range betweenany to the values.

X6. A device for reducing the time to add a reagent stored on a platesurface into a sample, as illustrated in FIGS. 1-4 and 15 c, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations, wherein the first        plate has, on its surface, a storage site that contains reagents        to be added into a sample, the reagents are capable of being        dissolving into the sample and diffusing in the sample; and        wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the transfer medium deposition in the        open configuration; and in the closed configuration: the plates        are facing each other, the spacers, the storage site, and at        least a portion of the sample are between the plates, the sample        contacts at least a portion of the storage site, the thickness        of the sample on the storage site is regulated by the plates and        the spacers, is thinner than the maximum thickness of the sample        when the plates are in the open configuration;    -   wherein the reduced thickness of the sample reduces the time for        mixing the reagents on the storage site with the sample.

In the method or the devices of any of paragraphs X1-6, in someembodiments, the relevant volume of the sample is the volume of thesample that sits on (i.e. on top of) the binding site or the storagesite.

In the method or the devices of any of paragraphs X1-6, in someembodiments, the relevant volume of the sample is the volume of thesample that sits on (i.e. on top of) the entire area or a partial areaof the binding site or the storage site.

In the method or the devices of any of paragraphs X1-6, in someembodiments, the ratio of the lateral dimension of the binding site orthe storage site to the sample thickness at the closed configuration is1.5 3 or larger, 3 or larger, 5 or larger, 10 or larger, 20 or larger,30 or larger, 50 or larger, 100 or larger, 200 or larger, 1000 orlarger, 10,000 or larger, or a range between any two of the values.

In the method or the devices of any of paragraphs X1-6, the ratio of thelateral dimension of the binding site or the storage site to the samplethickness at the closed configuration is between 3 and 20 in a preferredembodiment, 20 and 100 in another preferred embodiment, and 100 and 1000in another preferred embodiment, and 1000 and 10,000 in anotherpreferred embodiment.

In the method of any of paragraphs X1 and X3, in some embodiments, thefinal reduced sample thickness is significantly smaller than that of thearea of the binding site, so that the entity in the sample area that isoutside of the binding site will take longer time to bind to the bindingsite. With a proper selection of the incubation time, the entity thatbind to the binding sites will be primarily the entity in the samplevolume that sites on the binding site (i.e. the sample volume that isjust above the binding area). Then the calculation of the concentrationof the entity in the sample would be based on the sample thickness andthe binding site area.

In the method of paragraph X5, in some embodiments, the final reducedsample thickness is significantly smaller than that of the area of thestorage site, so that the entity

In the sample area that is outside of the binding site will take longertime to bind to the binding site. With a proper selection of theincubation time, the entity that bind to the binding sites will beprimarily the entity in the sample volume that sites on the binding site(i.e. the sample volume that is just above the binding area). Then thecalculation of the concentration of the entity in the sample would bebased on the sample thickness and the binding site area.

In the method of any of paragraphs X2, X4, X6, it further comprises acompressing device that bring the plates from an open configurations toa closed configurations. In some embodiments, the compressing device isone or any combination of the embodiments described in the disclosures

In the method of any of paragraphs X2, X4, X6, it further comprises acompressing device that bring the plates from an open configurations toa closed configurations, and a holding device that is configured to holdthe plates are in the closed configuration. In some embodiments, theholding device is one or any combination of the embodiments described inthe disclosures.

In the method of any of paragraphs X2, X4, X6, it further comprises acompressing device that bring the plates from an open configurations toa closed configurations, and a holding device that is configured to holdthe plates are in the closed configuration for a time of 0.001 sec orless, 0.01 sec or less, 0.1 sec or less, 1 sec or less, 5 sec or less,10 sec or less, 20 sec or less, 30 sec or less, 40 sec or less, 1 min orless, 2 min or less, 3 min or less, 5 min or less, 10 min or less, 20min or less, 30 min or less, 60 min or less, 90 min or less, 120 min orless, 180 min or less, 250 min or less, or a range between any two ofthese values.

In the method of any of paragraphs X2, X4, X6, it further comprises acompressing device that bring the plates from an open configurations toa closed configurations, and a holding device that is configured to holdthe plates are in the closed configuration for a time of, in a preferredembodiment, 0.001 sec or less, 0.01 sec or less, 0.1 sec or less, 1 secor less, 5 sec or less, 10 sec or less, 20 sec or less, 30 sec or less,40 sec or less, 1 min or less, 2 min or less, 3 min or less, or a rangebetween any two of these values.

Incubation Time.

In the method of any of paragraphs X1 and X3, it further comprises astep that, after (d) and while the plates are in the closedconfiguration, incubating for a time equal or less than a factor timesthe diffusion time of the entity in the sample diffusing across thesample thickness regulated by the plates at the closed configuration,and then stopping the incubation; wherein the incubation allows bindingof the entity to the binding site; and wherein the factor is 0.0001,0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100, 1000,10,000, or a range between any to the values. For example, if the factoris 1.1 and the diffusion time is 20 seconds, then the incubation time isequal to or less than 22 second. In one preferred embodiment, the factoris 0.1, 1, 1.5 or a range between any to the values.

In the method of paragraphs X5, it further comprises a step that, after(d) and while the plates are in the closed configuration, incubating fora time equal or less than a factor times the diffusion time of thereagents diffusing across the sample thickness regulated by the platesat the closed configuration, and then stopping the incubation; whereinthe incubation allows binding of the entity to the binding site; andwherein the factor is 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5,2, 3, 4, 5, 10, 100, 1000, 10,000, or a range between any to the values.For example, if the factor is 1.1 and the diffusion time is 20 seconds,then the incubation time is equal to or less than 22 second. In onepreferred embodiment, the factor is 0.1, 1, 1.5 or a range between anyto the values.

The method of any of paragraphs of X1, X3 and X5, or the device of anyof paragraph of X2, X4, and X6, wherein at least one of the spacers isinside the sample contact area.

The method of any of paragraphs of X1, X3 and X5, or the device of anyof paragraph of X2, X4, and X6, wherein spacers that have apredetermined inter-spacer distance.

In the method of any of paragraphs X1, X3, X5, it further comprises astep of incubation while the plates are in the closed configuration, thesaturation incubation time is 0.001 sec or less, 0.01 sec or less, 0.1sec or less, 1 sec or less, 5 sec or less, 10 sec or less, 20 sec orless, 30 sec or less, 40 sec or less, 1 min or less, 2 min or less, 3min or less, 5 min or less, 10 min or less, 20 min or less, 30 min orless, 60 min or less, 90 min or less, 120 min or less, 180 min or less,250 min or less, or a range between any two of these values.

In the method of any of paragraphs X1, X3, X5, the saturation incubationtime at the reduced sample thickness at the closed configuration is0.001 sec or less, 0.01 sec or less, 0.1 sec or less, 1 sec or less, 5sec or less, 10 sec or less, 20 sec or less, 30 sec or less, 40 sec orless, 1 min or less, 2 min or less, 3 min or less, 5 min or less, 10 minor less, 20 min or less, 30 min or less, 60 min or less, 90 min or less,120 min or less, 180 min or less, 250 min or less, or a range betweenany two of these values.

In some embodiments, capture agents are first immobilized at the bindingsite, then the sample are in contact with the binding site and theentity in the sample are captured by the capture agents, and finallydetection agents are added to be bound with the captured entity and thea signal from the detection agents will be read (e.g. by optical methodsor electrical methods or a combination). In some embodiments, otherreagents besides of capture agents and detection agents are added (e.g.blocking agent).

In many applications such as PoC, it is desirable to have simple and/orlow-cost devices and methods to add additional reagents into a sample.One aspect of the present invention is related to simple and/or low-costdevices and methods to add additional reagents into a sample. The addedadditional reagents include detection agents, blocking agents, lightsignal enhancers, light signal quenchers, or others. In some embodimentsof the present invention, it controls the assay processes by usingdifferent release time of the reagents stored on the same location. Thedifferent release time can be attached by adding other materials thathave different dissolve rate.

In certain embodiments, the reagent concentration mixed in the samplecan be controlled by controlling the sample thickness (e.g. control theratio of the sample thickness to the storage site area and/or the mixingtime).

2. Plates, Spacers, Scale-Marks, Sample Thickness Regulation 2.1 PlateConfigurations and Sample Thickness Regulation Open Configuration.

In some embodiments, in the open configuration, the two plates (i.e. thefirst plate and the second plate) are separated from each other. Incertain embodiments, the two plates have one side connected togetherduring all operations of the plates (including the open and closedconfiguration), the two plates open and close similar to a book. In someembodiments, the two plates have rectangle (or square) shape and havetwo sides of the rectangle connected together during all operations ofthe plates.

In some embodiments, the open configuration comprises a configurationthat the plates are far away from each other, so that the sample isdeposited onto one plate of the pair without a hindrance of the otherplate of the pair.

In some embodiments, the open configuration comprises a configurationthat the plates are far way, so that the sample is directly depositedonto one plate, as if the other plate does not exist.

In some embodiments, the open configuration comprises a configurationthat the pair of the plates are spaced apart by a distance at least 10nm, at least 100 nm, at least 1000 nm, at least 0.01 cm, at least 0.1cm, at least 0.5 cm, at least 1 cm, at least 2 cm, or at least 5 cm, ora range of any two of the values.

In some embodiments, the open configuration comprises a configurationthat the pair of plates are oriented in different orientations. In someembodiments, the open configuration comprises a configuration thatdefines an access gap between the pair of plates that is configured topermit sample addition.

In some embodiments, the open configuration comprises a configuration,wherein each plate has a sample contact surface and wherein at least oneof the contact surfaces of the plates is exposed when the plates are inthe one open configuration.

Closed Configuration and Sample Thickness Regulation.

In present invention, a closed configuration of the two plates is theconfiguration that a spacing (i.e. the distance) between the innersurfaces of the two plates is regulated by the spacers between the twoplates. Since the inner surfaces (also termed “sample surface”) of theplates are in contact with the sample during the compression step of aCROF process, hence at the closed configuration, the sample thickness isregulated by the spacers.

During the process of bring the plates from an open configuration to aclosed configuration, the plates are facing each other (at least a partof the plates are facing each other) and a force is used to bring thetwo plates together. When the two plates are brought from an openconfiguration to a closed configuration, the inner surfaces of the twoplate compress the sample deposited on the plate(s) to reduce the samplethickness (while the sample has an open flow laterally between theplates), and the thickness of a relevant volume of the sample isdetermined by the spacers, the plates, and the method being used and bythe sample mechanical/fluidic property. The thickness at a closedconfiguration can be predetermined for a given sample and given spacers,plates and plate pressing method.

The term “regulation of the spacing between the inner surfaces of theplates by the spacers” or “the regulation of the sample thickness by theplates and the spacer”, or a thickness of the sample is regulated by thespacers and the plates” means that the thickness of the sample in a CROFprocess is determined by a given plates, spacers, sample, and pressingmethod.

In some embodiments, the regulated sample thickness at the closedconfiguration is the same as the height of a spacer; in this case, atthe closed configuration, the spacers directly contact both plates(wherein one plate is the one that the spacer is fixed on, and the otherplate is the plate that is brought to contact with the spacer).

In certain embodiments, the regulated sample thickness at the closedconfiguration is larger than the height of a spacer, in this case, atthe closed configuration, the spacers directly contacts only the platethat has the spacers fixed or attached on its surface, and indirectlycontact the other plate (i.e. indirect contact). The term “indirectcontact” with a plate means that the spacer and the plate is separatedby a thin sample layer, which is termed “residual sample layer” and itsthickness is termed “the residue thickness”. For given spacers andplates, a given plate pressing method, and a given sample, the residualthickness can be predetermined (predetermined means prior to reach theclosed configuration), leading to a predetermination of the samplethickness at the closed configuration. This is because the residue layerthickness is the same for the given conditions (the sample, spacers,plates, and pressing force) and can be pre-calibrated and/or calculated.The regulated sample thickness is approximately equal to the spacerheight plus the sample residue thickness.

In many embodiments, the size and shape of the pillars arepre-characterized (i.e. pre-determined) before their use. And thepre-determined information are used to for later assaying, such asdetermination of the sample volume (or relevant volume) and others.

In some embodiments, the regulating of the sample thickness includesapplying a closing (compression) force to the plates to maintain thespacing between the plates.

In some embodiments, the regulating of the sample thickness includesestablishing the spacing between the plates with the spacers, a closingforce applied to the plates, and physical properties of the sample, andoptionally wherein the physical properties of the sample include atleast one of viscosity and compressibility.

2.2 Plates

In present invention, generally, the plates of CROF are made of anymaterial that (i) is capable of being used to regulate, together withthe spacers, the thickness of a portion or entire volume of the sample,and (ii) has no significant adverse effects to a sample, an assay, or agoal that the plates intend to accomplish. However, in certainembodiments, particular materials (hence their properties) are used forthe plate to achieve certain objectives.

In some embodiments, the two plates have the same or differentparameters for each of the following parameters: plate material, platethickness, plate shape, plate area, plate flexibility, plate surfaceproperty, and plate optical transparency.

Plate Materials.

The plates are made a single material, composite materials, multiplematerials, multilayer of materials, alloys, or a combination thereof.Each of the materials for the plate is an inorganic material, am organicmaterial, or a mix, wherein examples of the materials are given inparagraphs of Mat-1 and Mat-2.

Mat-1. The inorganic materials for the plates include, not limited to,glass, quartz, oxides, silicon-dioxide, silicon-nitride, hafnium oxide(HfO), aluminum oxide (AlO), semiconductors: (silicon, GaAs, GaN, etc.),metals (e.g. gold, silver, copper, aluminum, Ti, Ni, etc.), ceramics, orany combinations of thereof.Mat-2 The organic materials for the spacers include, not limited to,polymers (e.g. plastics) or amorphous organic materials. The polymermaterials for the spacers include, not limited to, acrylate polymers,vinyl polymers, olefin polymers, cellulosic polymers, noncellulosicpolymers, polyester polymers, Nylon, cyclic olefin copolymer (COC),poly(methyl methacrylate) (PMMA), polycarbonate (PC), cyclic olefinpolymer (COP), liquid crystalline polymer (LCP), polyamide (PA),polyethylene (PE), polyimide (PI), polypropylene (PP), poly(phenyleneether) (PPE), polystyrene (PS), polyoxymethylene (POM), polyether etherketone (PEEK), polyether sulfone (PES), poly(ethylene phthalate) (PET),polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidenefluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylenepropylene (FEP), perfluoroalkoxyalkane (PFA), polydimethylsiloxane(PDMS), rubbers, or any combinations of thereof.

In some embodiments, the plates are each independently made of at leastone of glass, plastic, ceramic, and metal. In some embodiments, eachplate independently includes at least one of glass, plastic, ceramic,and metal.

In some embodiments, one plate is different from the other plate inlateral area, thickness, shape, materials, or surface treatment. In someembodiments, one plate is the same as the other plate in lateral area,thickness, shape, materials, or surface treatment.

The materials for the plates are rigid, flexible or any flexibilitybetween the two. The rigid (i.e. stiff) or flexibility is relative to agive pressing forces used in bringing the plates into the closedconfiguration.

In some embodiments, a selection of rigid or flexible plate aredetermined from the requirements of controlling a uniformity of thesample thickness at the closed configuration.

In some embodiments, at least one of the two plates are transparent (toa light). In some embodiments at least a part or several parts of oneplate or both plates are transparent. In some embodiments, the platesare non-transparent.

Plate Thickness.

In some embodiments, the average thicknesses for at least one of theplates are 2 nm or less, 10 nm or less, 100 nm or less, 500 nm or less,1000 nm or less, 2 um (micron) or less, 5 um or less, 10 um or less, 20um or less, 50 um or less, 100 um or less, 150 um or less, 200 um orless, 300 um or less, 500 um or less, 800 um or less, 1 mm (millimeter)or less, 2 mm or less, 3 mm or less, or a range between any two of thevalues.

In some embodiments, the average thicknesses for at least one of theplates are at most 3 mm (millimeter), at most 5 mm, at most 10 mm, atmost 20 mm, at most 50 mm, at most 100 mm, at most 500 mm, or a rangebetween any two of the values.

In some embodiments, the thickness of a plate is not uniform across theplate. Using a different plate thickness at different location can beused to control the plate bending, folding, sample thickness regulation,and others.

Plate Shape and Area.

Generally, the plates can have any shapes, as long as the shape allows acompress open flow of the sample and the regulation of the samplethickness. However, in certain embodiments, a particular shape may beadvantageous. The shape of the plate can be round, elliptical,rectangles, triangles, polygons, ring-shaped, or any superpositions ofthese shapes.

In some embodiments, the two plates can have the same size or shape, ordifferent. The area of the plates depend on the application. The area ofthe plate is at most 1 mm2 (millimeter square), at most 10 mm2, at most100 mm2, at most 1 cm2 (centimeter square), at most 5 cm2, at most 10cm2, at most 100 cm2, at most 500 cm2, at most 1000 cm2, at most 5000cm2, at most 10,000 cm2, or over 10,000 cm2, or any arrange between anyof the two values. The shape of the plate can be rectangle, square,round, or others.

In certain embodiments, at least one of the plate is in the form of abelt (or strip) that has a width, thickness, and length. The width is atmost 0.1 cm (centimeter), at most 0.5 cm, at most 1 cm, at most 5 cm, atmost 10 cm, at most 50 cm, at most 100 cm, at most 500 cm, at most 1000cm, or a range between any two of the values. The length can be as longit needed. The belt can be rolled into a roll.

Plate Surface Flatness.

In many embodiments, an inner surface of the plates are flat orsignificantly flat, planar. In certain embodiments, the two innersurfaces are, at the closed configuration, parallel with each other.Flat inner surfaces facilitates a quantification and/or controlling ofthe sample thickness by simply using the predetermined spacer height atthe closed configuration. For non-flat inner surfaces of the plate, oneneed to know not only the spacer height, but also the exact the topologyof the inner surface to quantify and/or control the sample thickness atthe closed configuration. To know the surface topology needs additionalmeasurements and/or corrections, which can be complex, time consuming,and costly.

A flatness of the plate surface is relative to the final samplethickness (the final thickness is the thickness at the closedconfiguration), and is often characterized by the term of “relativesurface flatness” is the ratio of the plate surface flatness variationto the final sample thickness.

In some embodiments, the relative surface is less than 0.01%, 0.1%, lessthan 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, lessthan 20%, less than 30%, less than 50%, less than 70%, less than 80%,less than 100%, or a range between any two of these values.

Plate Surface Parallelness.

In some embodiments, the two surfaces of the plate is significantlyparallel with each other. In certain embodiments, the two surfaces ofthe plate is not parallel with each other.

Plate Flexibility.

In some embodiments, a plate is flexible under the compressing of a CROFprocess. In some embodiments, both plates are flexible under thecompressing of a CROF process. In some embodiments, a plate is rigid andanother plate is flexible under the compressing of a CROF process. Insome embodiments, both plates are rigid. In some embodiments, both plateare flexible but have different flexibility.

Plate Optical Transparency.

In some embodiments, a plate is optical transparent. In someembodiments, both plates are optical transparent. In some embodiments, aplate is optical transparent and another plate is opaque. In someembodiments, both plates are opaque. In some embodiments, both plate areoptical transparent but have different optical transparency. The opticaltransparency of a plate refers a part or the entire area of the plate.

Surface Wetting Properties.

In some embodiments, a plate has an inner surface that wets (i.e.contact angle is less 90 degree) the sample, the transfer liquid, orboth. In some embodiments, both plates have an inner surface that wetsthe sample, the transfer liquid, or both; either with the same ordifferent wettability. In some embodiments, a plate has an inner surfacethat wets the sample, the transfer liquid, or both; and another platehas an inner surface that does not wet (i.e. the contact angle equal toor larger than 90 degree). The wetting of a plate inner surface refers apart or the entire area of the plate.

In some embodiments, the inner surface of the plate has other nano ormicrostructures to control a lateral flow of a sample during a CROF. Thenano or microstructures include, but not limited to, channels, pumps,and others. Nano and microstructures are also used to control thewetting properties of an inner surface.

2.3 Spacers

Spacers' Function.

In present invention, the spacers are configured to have one or anycombinations of the following functions and properties: the spacers areconfigured to (1) control, together with the plates, the thickness ofthe sample or a relevant volume of the sample (Preferably, the thicknesscontrol is precise, or uniform or both, over a relevant area); (2) allowthe sample to have a compressed regulated open flow (CROF) on platesurface; (3) not take significant surface area (volume) in a givensample area (volume); (4) reduce or increase the effect of sedimentationof particles or analytes in the sample; (5) change and/or control thewetting propertied of the inner surface of the plates; (6) identify alocation of the plate, a scale of size, and/or the information relatedto a plate, or (7) do any combination of the above.

Spacer Architectures and Shapes.

To achieve desired sample thickness reduction and control, in certainembodiments, the spacers are fixed its respective plate. In general, thespacer can have any shape, as long as the spacers are capable ofregulating the sample thickness during a CROF process, but certainshapes are preferred to achieve certain functions, such as betteruniformity, less overshoot in pressing, etc.

The spacer(s) is a single spacer or a plurality of spacers. (e.g. anarray). Some embodiments of a plurality of spacers is an array ofspacers (e.g. pillars), where the inter-spacer distance is periodic oraperiodic, or is periodic or aperiodic in certain areas of the plates,or has different distances in different areas of the plates.

There are two kinds of the spacers: open-spacers and enclosed-spacers.The open-spacer is the spacer that allows a sample to flow through thespacer (i.e. the sample flows around and pass the spacer. For example, apost as the spacer), and the enclosed spacer is the spacer that stop thesample flow (i.e. the sample cannot flow beyond the spacer. For example,a ring shape spacer and the sample is inside the ring.). Both types ofspacers use their height to regular the final sample thickness at aclosed configuration.

In some embodiments, the spacers are open-spacers only. In someembodiments, the spacers are enclosed-spacers only. In some embodiments,the spacers are a combination of open-spacers and enclosed-spacers.

The term “pillar spacer” means that the spacer has a pillar shape andthe pillar shape refers to an object that has height and a lateral shapethat allow a sample to flow around it during a compressed open flow.

In some embodiments, the lateral shapes of the pillar spacers are theshape selected from the groups of (i) round, elliptical, rectangles,triangles, polygons, ring-shaped, star-shaped, letter-shaped (e.g.L-shaped, C-shaped, the letters from A to Z), number shaped (e.g. theshapes like 0 1, 2, 3, 4, . . . to 9); (ii) the shapes in group (i) withat least one rounded corners; (iii) the shape from group (i) withzig-zag or rough edges; and (iv) any superposition of (i), (ii) and(iii). For multiple spacers, different spacers can have differentlateral shape and size and different distance from the neighboringspacers.

In some embodiments, the spacers may be and/or may include posts,columns, beads, spheres, and/or other suitable geometries. The lateralshape and dimension (i.e., transverse to the respective plate surface)of the spacers can be anything, except, in some embodiments, thefollowing restrictions: (i) the spacer geometry will not cause asignificant error in measuring the sample thickness and volume; or (ii)the spacer geometry would not prevent the out-flowing of the samplebetween the plates (i.e. it is not in enclosed form). But in someembodiments, they require some spacers to be closed spacers to restrictthe sample flow.

In some embodiments, the shapes of the spacers have rounded corners. Forexample, a rectangle shaped spacer has one, several or all cornersrounded (like a circle rather 90 degree angle). A round corner oftenmake a fabrication of the spacer easier, and in some cases less damageto a biological material.

The sidewall of the pillars can be straight, curved, sloped, ordifferent shaped in different section of the sidewall. In someembodiments, the spacers are pillars of various lateral shapes,sidewalls, and pillar-height to pillar lateral area ratio.

In a preferred embodiment, the spacers have shapes of pillars forallowing open flow.

Spacers' Materials.

In the present invention, the spacers are generally made of any materialthat is capable of being used to regulate, together with the two plates,the thickness of a relevant volume of the sample. In some embodiments,the materials for the spacers are different from that for the plates. Insome embodiments, the materials for the spaces are at least the same asa part of the materials for at least one plate.

The spacers are made a single material, composite materials, multiplematerials, multilayer of materials, alloys, or a combination thereof.Each of the materials for the spacers is an inorganic material, amorganic material, or a mix, wherein examples of the materials are givenin paragraphs of Mat-1 and Mat-2. In a preferred embodiment, the spacersare made in the same material as a plate used in CROF.

Spacer's Mechanical Strength and Flexibility.

In some embodiments, the mechanical strength of the spacers are strongenough, so that during the compression and at the closed configurationof the plates, the height of the spacers is the same or significantlysame as that when the plates are in an open configuration. In someembodiments, the differences of the spacers between the openconfiguration and the closed configuration can be characterized andpredetermined.

The material for the spacers is rigid, flexible or any flexibilitybetween the two. The rigid is relative to a give pressing forces used inbringing the plates into the closed configuration: if the space does notdeform greater than 1% in its height under the pressing force, thespacer material is regarded as rigid, otherwise a flexible. When aspacer is made of material flexible, the final sample thickness at aclosed configuration still can be predetermined from the pressing forceand the mechanical property of the spacer.

Spacer inside Sample.

To achieve desired sample thickness reduction and control, particularlyto achieve a good sample thickness uniformity, in certain embodiments,the spacers are placed inside the sample, or the relevant volume of thesample. In some embodiments, there are one or more spacers inside thesample or the relevant volume of the sample, with a proper inter spacerdistance. In certain embodiments, at least one of the spacers is insidethe sample, at least two of the spacers inside the sample or therelevant volume of the sample, or at least of “n” spacers inside thesample or the relevant volume of the sample, where “n” may be determinedby a sample thickness uniformity or a required sample flow propertyduring a CROF.

Spacer Height.

In some embodiments, all spacers have the same pre-determined height. Insome embodiments, spacers have different pre-determined height. In someembodiments, spacers can be divided into groups or regions, wherein eachgroup or region has its own spacer height. And in certain embodiments,the predetermined height of the spacers is an average height of thespacers. In some embodiments, the spacers have approximately the sameheight. In some embodiments, a percentage of number of the spacers havethe same height.

The height of the spacers is selected by a desired regulated finalsample thickness and the residue sample thickness. The spacer height(the predetermined spacer height) and/or sample thickness is 3 nm orless, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500nm or less, 800 nm or less, 1000 nm or less, 1 um or less, 2 um or less,3 um or less, 5 um or less, 10 um or less, 20 um or less, 30 um or less,50 um or less, 100 um or less, 150 um or less, 200 um or less, 300 um orless, 500 um or less, 800 um or less, 1 mm or less, 2 mm or less, 4 mmor less, or a range between any two of the values.

The spacer height and/or sample thickness is between 1 nm to 100 nm inone preferred embodiment, 100 nm to 500 nm in another preferredembodiment, 500 nm to 1000 nm in a separate preferred embodiment, 1 um(i.e. 1000 nm) to 2 um in another preferred embodiment, 2 um to 3 um ina separate preferred embodiment, 3 um to 5 um in another preferredembodiment, 5 um to 10 um in a separate preferred embodiment, and 10 umto 50 um in another preferred embodiment, 50 um to 100 um in a separatepreferred embodiment.

In some embodiments, the spacer height and/or sample thickness (i) equalto or slightly larger than the minimum dimension of an analyte, or (ii)equal to or slightly larger than the maximum dimension of an analyte.The “slightly larger” means that it is about 1% to 5% larger and anynumber between the two values.

In some embodiments, the spacer height and/or sample thickness is largerthan the minimum dimension of an analyte (e.g. an analyte has ananisotropic shape), but less than the maximum dimension of the analyte.

For example, the red blood cell has a disk shape with a minim dimensionof 2 um (disk thickness) and a maximum dimension of 11 um (a diskdiameter). In an embodiment of the present invention, the spacers isselected to make the inner surface spacing of the plates in a relevantarea to be 2 um (equal to the minimum dimension) in one embodiment, 2.2um in another embodiment, or 3 (50% larger than the minimum dimension)in other embodiment, but less than the maximum dimension of the redblood cell. Such embodiment has certain advantages in blood cellcounting. In one embodiment, for red blood cell counting, by making theinner surface spacing at 2 or 3 um and any number between the twovalues, a undiluted whole blood sample is confined in the spacing, onaverage, each red blood cell (RBC) does not overlap with others,allowing an accurate counting of the red blood cells visually. (Too manyoverlaps between the RBC's can cause serious errors in counting).

In the present invention, in some embodiments, it uses the plates andthe spacers to regulate not only a thickness of a sample, but also theorientation and/or surface density of the analytes/entity in the samplewhen the plates are at the closed configuration. When the plates are ata closed configuration, a thinner thickness of the sample gives a lessthe analytes/entity per surface area (i.e. less surface concentration).

Spacer Lateral Dimension.

For an open-spacer, the lateral dimensions can be characterized by itslateral dimension (sometime being called width) in the x and y-twoorthogonal directions. The lateral dimension of a spacer in eachdirection is the same or different. In some embodiments, the lateraldimension for each direction (x or y) is . . . .

In some embodiments, the ratio of the lateral dimensions of x to ydirection is 1, 1.5, 2, 5, 10, 100, 500, 1000, 10,000, or a rangebetween any two of the value. In some embodiments, a different ratio isused to regulate the sample flow direction; the larger the ratio, theflow is along one direction (larger size direction).

In some embodiments, the different lateral dimensions of the spacers inx and y direction are used as (a) using the spacers as scale-markers toindicate the orientation of the plates, (b) using the spacers to createmore sample flow in a preferred direction, or both.

In a preferred embodiment, the period, width, and height.

In some embodiments, all spacers have the same shape and dimensions. Insome embodiments, each spacers have different lateral dimensions.

For enclosed-spacers, in some embodiments, the inner lateral shape andsize are selected based on the total volume of a sample to be enclosedby the enclosed spacer(s), wherein the volume size has been described inthe present disclosure; and in certain embodiments, the outer lateralshape and size are selected based on the needed strength to support thepressure of the liquid against the spacer and the compress pressure thatpresses the plates.

Aspect Ratio of Height to the Average Lateral Dimension of PillarSpacer.

In certain embodiments, the aspect ratio of the height to the averagelateral dimension of the pillar spacer is 100,000, 10,000, 1,000, 100,10, 1, 0.1, 0.01, 0.001, 0.0001, 0, 00001, or a range between any two ofthe values.

Spacer Height Precisions.

The spacer height should be controlled precisely. The relative precisionof the spacer (i.e. the ratio of the deviation to the desired spacerheight) is 0.001% or less, 0.01% or less, 0.1% or less; 0.5% or less, 1%or less, 2% or less, 5% or less, 8% or less, 10% or less, 15% or less,20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% orless, 80% or less, 90% or less, 99.9% or less, or a range between any ofthe values.

Inter-Spacer Distance.

The spacers can be a single spacer or a plurality of spacers on theplate or in a relevant area of the sample. In some embodiments, thespacers on the plates are configured and/or arranged in an array form,and the array is a periodic, non-periodic array or periodic in somelocations of the plate while non-periodic in other locations.

In some embodiments, the periodic array of the spacers has a lattice ofsquare, rectangle, triangle, hexagon, polygon, or any combinations ofthereof, where a combination means that different locations of a platehas different spacer lattices.

In some embodiments, the inter-spacer distance of a spacer array isperiodic (i.e. uniform inter-spacer distance) in at least one directionof the array. In some embodiments, the inter-spacer distance isconfigured to improve the uniformity between the plate spacing at aclosed configuration.

The distance between neighboring spacers (i.e. the inter-spacerdistance) is 1 um or less, 5 um or less, 10 um or less, 20 um or less,30 um or less, 40 um or less, 50 um or less, 60 um or less, 70 um orless, 80 um or less, 90 um or less, 100 um or less, 200 um or less, 300um or less, 400 um or less, or a range between any two of the values.

In certain embodiments, the inter-spacer distance is at 400 or less, 500or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 7 mm orless, 10 mm or less, or any range between the values. In certainembodiments, the inter-spacer distance is al0 mm or less, 20 mm or less,30 mm or less, 50 mm or less, 70 mm or less, 100 mm or less, or anyrange between the values.

The distance between neighboring spacers (i.e. the inter-spacerdistance) is selected so that for a given properties of the plates and asample, at the closed-configuration of the plates, the sample thicknessvariation between two neighboring spacers is, in some embodiments, atmost 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or any range between thevalues; or in certain embodiments, at most 80%, 100%, 200%, 400%, or arange between any two of the values.

Clearly, for maintaining a given sample thickness variation between twoneighboring spacers, when a more flexible plate is used, a closerinter-spacer distance is needed.

Specify the Accuracy of the Inter Spacer Distance.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

The period of spacer array is between 1 nm to 100 nm in one preferredembodiment, 100 nm to 500 nm in another preferred embodiment, 500 nm to1000 nm in a separate preferred embodiment, 1 um (i.e. 1000 nm) to 2 umin another preferred embodiment, 2 um to 3 um in a separate preferredembodiment, 3 um to 5 um in another preferred embodiment, 5 um to 10 umin a separate preferred embodiment, and 10 um to 50 um in anotherpreferred embodiment, 50 um to 100 um in a separate preferredembodiment, 100 um to 175 um in a separate preferred embodiment, and 175um to 300 um in a separate preferred embodiment.

Spacer Density.

The spacers are arranged on the respective plates at a surface densityof greater than one per um², greater than one per 10 um², greater thanone per 100 um², greater than one per 500 um², greater than one per 1000um², greater than one per 5000 um², greater than one per 0.01 mm²,greater than one per 0.1 mm², greater than one per 1 mm², greater thanone per 5 mm², greater than one per 10 mm², greater than one per 100mm², greater than one per 1000 mm², greater than one per 10000 mm², or arange between any two of the values.

(3) the spacers are configured to not take significant surface area(volume) in a given sample area (volume);

Ratio of Spacer Volume to Sample Volume.

In many embodiments, the ratio of the spacer volume (i.e. the volume ofthe spacer) to sample volume (i.e. the volume of the sample), and/or theratio of the volume of the spacers that are inside of the relevantvolume of the sample to the relevant volume of the sample are controlledfor achieving certain advantages. The advantages include, but notlimited to, the uniformity of the sample thickness control, theuniformity of analytes, the sample flow properties (i.e. flow speed,flow direction, etc.).

In certain embodiments, the ratio of the spacer volume r) to samplevolume, and/or the ratio of the volume of the spacers that are inside ofthe relevant volume of the sample to the relevant volume of the sampleis less than 100%, at most 99%, at most 70%, at most 50%, at most 30%,at most 10%, at most 5%, at most 3% at most 1%, at most 0.1%, at most0.01%, at most 0.001%, or a range between any of the values.

Spacers Fixed to Plates.

The inter spacer distance and the orientation of the spacers, which playa key role in the present invention, are preferably maintained duringthe process of bringing the plates from an open configuration to theclosed configuration, and/or are preferably predetermined before theprocess from an open configuration to a closed configurations.

Some embodiments of the present invention is that the spacers are fixedon one of the plates before bring the plates to the closedconfiguration. The term “a spacer is fixed with its respective plate”means that the spacer is attached to a plate and the attachment ismaintained during a use of the plate. An example of “a spacer is fixedwith its respective plate” is that a spacer is monolithically made ofone piece of material of the plate, and the position of the spacerrelative to the plate surface does not change. An example of “a spaceris not fixed with its respective plate” is that a spacer is glued to aplate by an adhesive, but during a use of the plate, the adhesive cannothold the spacer at its original location on the plate surface (i.e. thespacer moves away from its original position on the plate surface).

In some embodiments, at least one of the spacers are fixed to itsrespective plate. In certain embodiments, at two spacers are fixed toits respective plates. In certain embodiments, a majority of the spacersare fixed with their respective plates. In certain embodiments, all ofthe spacers are fixed with their respective plates.

In some embodiments, a spacer is fixed to a plate monolithically.

In some embodiments, the spacers are fixed to its respective plate byone or any combination of the following methods and/or configurations:attached to, bonded to, fused to, imprinted, and etched.

The term “imprinted” means that a spacer and a plate are fixedmonolithically by imprinting (i.e. embossing) a piece of a material toform the spacer on the plate surface. The material can be single layerof a material or multiple layers of the material.

The term “etched” means that a spacer and a plate are fixedmonolithically by etching a piece of a material to form the spacer onthe plate surface. The material can be single layer of a material ormultiple layers of the material.

The term “fused to” means that a spacer and a plate are fixedmonolithically by attaching a spacer and a plate together, the originalmaterials for the spacer and the plate fused into each other, and thereis clear material boundary between the two materials after the fusion.

The term “bonded to” means that a spacer and a plate are fixedmonolithically by binding a spacer and a plate by adhesion.

The term “attached to” means that a spacer and a plate are connectedtogether.

In some embodiments, the spacers and the plate are made in the samematerials. In other embodiment, the spacers and the plate are made fromdifferent materials. In other embodiment, the spacer and the plate areformed in one piece. In other embodiment, the spacer has one end fixedto its respective plate, while the end is open for accommodatingdifferent configurations of the two plates.

In other embodiment, each of the spacers independently is at least oneof attached to, bonded to, fused to, imprinted in, and etched in therespective plate. The term “independently” means that one spacer isfixed with its respective plate by a same or a different method that isselected from the methods of attached to, bonded to, fused to, imprintedin, and etched in the respective plate.

In some embodiments, at least a distance between two spacers ispredetermined (“predetermined inter-spacer distance” means that thedistance is known when a user uses the plates.).

In some embodiments of all methods and devices described herein, thereare additional spacers besides to the fixed spacers.

Specific Sample Thickness.

In present invention, it was observed that a larger plate holding force(i.e. the force that holds the two plates together) can be achieved byusing a smaller plate spacing (for a given sample area), or a largersample area (for a given plate-spacing), or both.

In some embodiments, at least one of the plates is transparent in aregion encompassing the relevant area, each plate has an inner surfaceconfigured to contact the sample in the closed configuration; the innersurfaces of the plates are substantially parallel with each other, inthe closed configuration; the inner surfaces of the plates aresubstantially planar, except the locations that have the spacers; or anycombination of thereof.

2.4 Final Sample Thickness and Uniformity

In some embodiments, significantly flat is determined relative to thefinal sample thickness, and has, depending upon on embodiments andapplications, a ratio of to the sample thickness of less than 0.1%, lessthan 0.5%, less than 1%, less than 2%, less than 5%, or less than 10%,or a range between any two of these values.

In some embodiments, flatness relative to the sample thickness may beless than 0.1%, less than 0.5%, less than 1%, less than 2%, less than5%, less than 10%, less than 20%, less than 50%, or less than 100%, or arange between any two of these values.

In some embodiments, significantly flat may mean that the surfaceflatness variation itself (measured from an average thickness) is lessthan 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, orless than 10%, or a range between any two of these values. Generally,flatness relative to the plate thickness may be less than 0.1%, lessthan 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, lessthan 20%, less than 50%, or less than 100%, or a range between any twoof these values.

2.5 Spacer Fabrication Methods.

The spacers can be fabricated on a plate in a variety of ways, usinglithography, etching, embossing (nanoimprint), depositions, lift-off,fusing, or a combination of thereof. In some embodiments, the spacersare directly embossed or imprinted on the plates. In some embodiments,the spacers imprinted into a material (e.g. plastics) that is depositedon the plates. In certain embodiments, the spacers are made by directlyembossing a surface of a CROF plate. The nanoimprinting may be done byroll to roll technology using a roller imprinter, or roll to a planarnanoimprint. Such process has a great economic advantage and hencelowering the cost.

In some embodiments, the spacers are deposited on the plates. Thedeposition can be evaporation, pasting, or a lift-off. In the pasting,the spacer is fabricated first on a carrier, then the spacer istransferred from the carrier to the plate. In the lift-off, a removablematerial is first deposited on the plate and holes are created in thematerial; the hole bottom expose the plate surface and then a spacermaterial is deposited into the hole and afterwards the removablematerial is removed, leaving only the spacers on the plate surface. Insome embodiments, the spacers deposited on the plate are fused with theplate. In some embodiments, the spacer and the plates are fabricated ina single process. The single process includes imprinting (i.e.embossing, molding) or synthesis.

In some embodiments, at least two of the spacers are fixed to therespective plate by different fabrication methods, and optionallywherein the different fabrication methods include at least one of beingdeposition, bonded, fuse, imprinted, and etched.

In some embodiments, one or more of the spacers are fixed to therespective plate(s) is by a fabrication method of being bonded, beingfused, being imprinted, or being etched, or any combination of thereof.

In some embodiments, the fabrication methods for forming such monolithicspacers on the plate include a method of being bonded, being fused,being imprinted, or being etched, or any combination of thereof.

2.6 Scale-Markers

The term “scale-marker(s) refers to the scale-marker(s) that able toassist a quantification (i.e. dimension measurement) or a control of therelevant area and/or the relative volume of a sample. In someembodiments, the scale-markers are on the first plate or the secondplate, on both on plates, on one surface of the plate, on both surfacesof the plate, between the plates, near the plates, or any combination ofthereof. In some embodiments, the scale-markers are fixed on the firstplate or the second plate, on both on plates, on one surface of theplate, on both surfaces of the plate, between the plates, near theplates, or any combination of thereof. In some embodiments, thescale-markers are deposited on the first plate or the second plate, onboth on plates, on one surface of the plate, on both surfaces of theplate, between the plates, near the plates, or any combination ofthereof. In some embodiments, some of spacers are fixed and some spacersare deposited.

In some embodiments, the scale-marks are etched scale-marks, depositedmaterials, or printed materials. In certain embodiments, the materialsthat absorbing the light, reflecting light, emitting light, or anycombination of thereof.

In some embodiments, the scale-markers are a or a plurality of object(s)with known dimensions and/or known separation distances. Examples of theobjects include, not limited to, rectangles, cylinders, or circles.

In some embodiments, the scale-markers have a dimension of in the rangeof nanometers (nm), microns (um) or millimeters (mm) or other sizes.

In some embodiments, the scale-markers are a ruler, which has scalescale-marks that are configured to measure a dimension of an object. Insome embodiments, the scale-marks are in the scale of nanometer (nm),microns (um) or millimeter (mm) or other sizes. In some embodiments, thescale marks are etched scale-marks, deposited materials, or printedmaterials. In some embodiments, the materials for the scale-markers arethe materials that absorbing the light, reflecting light, scatteringlight, interfering light, diffracting light, emitting light, or anycombination of thereof.

In some embodiments, the makers are the spacers, which server dualfunctions of “regulating sample thickness” and “providing scale-markingand/or dimension scaling”. For examples, a rectangle spacer with a knowndimension or two spacers with a known separation distance can be used tomeasure a dimension related to the sample round the spacer(s). From themeasured sample dimension, one can calculate the volume of the relevantvolume of the sample.

In some embodiments, the scale-markers is configured to at leastpartially define a boundary of the relevant volume of the sample.

In some embodiments, at least one of the scale-markers is configured tohave a known dimension that is parallel to a plane of the lateral areaof the relevant volume of the sample.

In some embodiments, at least a pair of the scale-markers are separatedby a known distance that is parallel to a plane of the lateral area.

In some embodiments, the scale-markers are configured for opticaldetection.

In some embodiments, each scale-marker independently is at least one oflight absorbing, light reflecting, light scattering, light diffracting,and light emitting.

In some embodiments, the scale-markers are arranged in a regular arraywith a known lateral spacing.

In some embodiments, each scale-marker independently has a lateralprofile that is at least one of square, rectangular, polygonal, andcircular.

In some embodiments, at least one of the scale-markers is attached to,bonded to, fused to, imprinted in, and etched in one of the plates.

In some embodiments, at least one of the scale-markers is one of thespacers.

In some embodiments, some spacers also play a role of scale-marker toquantification of a relevant volume of the sample.

In certain embodiments, a binding site(s) (that immobilizes theanalytes), storage sites, or alike, serves as a scale-marker(s). In oneembodiment, the site with a known lateral dimension interacts with lightgenerating a detectable signal, that reals the known lateral dimensionof the site, hence serving a scale-marker(s).

In another embodiment, the dimension of the sites are predeterminedbefore a CROF process and the thickness of the portion of the samplesitting on the site is, when the plates are at the closed configuration,significantly smaller than the lateral average dimension of the site,then by controlling the incubation time so that, after the incubation,(1) the majority of the analytes/entity that bind to the binding sitecome from the sample volume that sites on top of the binding site, or(2) the majority of the reagent that is mixed (diffused) into the samplevolume that sites on top of the binding site come from the storage site.In these cases, the relevant volume of the sample to the binding or thereagent mixing is the volume that is approximately equal to thepredetermined site area multiplies the sample thickness at the site. Akey reason for this be possible is that, for the given incubation time,the analytes/entity in the sample volume outside the relevant volume donot have enough time to diffuse into the binding site, or the reagentson the storage site do not have enough time to diffuse into in thesample volume outside the relevant volume.

An example to illustrate the method of measuring and/or controlling therelevant area and volume by using a site with known dimension and bylimiting the incubation time is that an assay has a binding site (i.e.the area with capture agents) of 1,000 um by 1000 um on a first plate ofa CROF process (which has a surface large than the binding site); at theclosed configuration of the plates, a sample with analytes is over thebinding site, has a thickness of about 20 um (in the bind site area) andan area larger than the binding site and is incubated for a time equalto the target analyte/entity diffusion time across the sample thickness.In this case, the majority of the analytes/entity that bind to thebinding site come from the sample volume that sites on top of thebinding site, which is 1,000 um by 1000 um by 20 um=0.02 p, because theanalytes in the sample portion that is 20 um away from the binding sitedo not have time to diffuse to the binding site (statistically). In thiscase, if the signal, due to the analytes/entity captured by the bindingsite, is measured after the incubation, one can determine theanalyte/entity concentration in the relevant area and relevant volume ofthe sample from the information (provided by the binding site) of therelevant area and relevant volume. The analyte concentration isquantified by the number of analytes captured by the binding sitedivided the relevant volume.

In some embodiments, the relevant volume is approximately equal to thebinding site area times the sample thickness, and the target analyteconcentration in the sample is approximately equal to the number ofanalyte captured by the binding site divided by the relevant samplevolume. This accuracy of the method of quantification of target analytevolume gets better as the ratio of the binding site dimension to thesample thickness gets larger (assuming the incubation time is about thetarget analyte diffusion time in the sample for a distance of the samplethickness).

Spreading Times in CROF.

In the present invention, in the methods and the devices of allparagraphs that spread the sample by two plates, the time for spreadingthe sample to the final thickness at a closed configuration is 0.001 secor less, 0.01 sec, 0.1 sec, 1 sec, 5 sec, 10 sec, 20 sec, 30 sec, 60sec, 90 sec, 100 sec, 150 sec, 200 sec, 300 sec, 500 sec, 1000 sec, or arange between any two of the values.

In the methods and the devices of all paragraphs that spread the sampleby two plates, in a preferred embodiment, the time for spreading thesample to the final thickness at a closed configuration is 0.001 sec orless, 0.01 sec, 0.1 sec, 1 sec, 3 sec, 5 sec, 10 sec, 20 sec, 30 sec, 60sec, 90 sec, 100 sec, 150 sec, or a range between any two of the values.

In the methods and the devices of all paragraphs that spread the sampleby two plates, in a preferred embodiment, the time for spreading thesample to the final thickness at a closed configuration is 0.001 sec orless, 0.01 sec, 0.1 sec, 1 sec, 3 sec, 5 sec, 10 sec, 20 sec, 30 sec, 60sec, 90 sec, or a range between any two of the values.

In the methods and the devices of all paragraphs that spread the sampleby two plates, in a preferred embodiment, the time for spreading thesample to the final thickness at a closed configuration is 0.001 sec orless, 0.01 sec, 0.1 sec, 1 sec, 3 sec, 5 sec, 10 sec, 20 sec, 30 sec, ora range between any two of the values.

In the methods and the devices of all paragraphs that spread the sampleby two plates, in a preferred embodiment, the time for spreading thesample to the final thickness at a closed configuration is 0.001 sec orless, 0.01 sec, 0.1 sec, 1 sec, 3 sec, 5 sec, 10 sec, or a range betweenany two of the values.

In the methods and the devices of all paragraphs that spread the sampleby two plates, in a preferred embodiment, the time for spreading thesample to the final thickness at a closed configuration is 0.001 sec orless, 0.01 sec, 0.1 sec, 1 sec, 3 sec, or a range between any two of thevalues.

The embodiments and any of their combinations described in the Section 3are applied to (i.e. are combined with) other embodiments in the entiredescription of the present invention.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, and the thickness of theX-Plate is from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, and the thickness of theX-Plate is from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate and are made of the same materials, and the thickness of theX-Plate is from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate a thin plastic film using a mold, and are made of the samematerials, and the thickness of the X-Plate is from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene).

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene) and thethickness of the X-Plate is from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene) and thethickness of the X-Plate is from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene), and thespacers have either a square or rectangle shape, and have the samespacer height.

In one preferred embodiment, the spacers have a square or rectangleshape (with or without round corners).

In one preferred embodiment, the spacers have square or rectanglepillars with the pillar width (spacer width in each lateral direction)between 1 um to 200 um; pillar period (i.e. spacer period) from 2um-2000 um, and pillar height (i.e. spacer height) from 1 um-100 um.

In one preferred embodiment, the spacers made of PMMA or PS have squareor rectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-100um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate and are made of plastic materials, and the spacers have squareor rectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-100um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate and are made of the same materials, and the spacers have squareor rectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-10um.

In one preferred embodiment, the spacers are monolithically made on theX-Plate and are made of the same materials selected from PS or PMMA orother plastics, and the spacers have square or rectangle pillars withthe pillar width (spacer width in each lateral direction) between 1 umto 200 um; pillar period (i.e. spacer period) from 2 um-2000 um, andpillar height (i.e. spacer height) from 10 um-50 um.

In one preferred embodiment of a CROF device, one plate is X-Plate andthe other plate is a planar thin film, wherein the thickness of at leastone of the plates is in a range of from 10 um to 250 um; wherein thespacers are fixed on the X-Plate, and wherein the plates and the spacerscan have the same materials or different materials and are made of PMMA(polymethyl methacrylate), PS (polystyrene), or a material of similarmechanical properties as PMMA or PS.

In one preferred embodiment of a CROF device, one plate is X-Plate andthe other plate is a planar thin film, wherein the thickness of at leastone of the plates is in a range of from 250 um to 500 um; wherein thespacers are fixed on the X-Plate, and wherein the plates and the spacerscan have the same materials or different materials and are made of PMMA(polymethyl methacrylate), PS (polystyrene), or a material of similarmechanical properties as PMMA or PS.

In one preferred embodiment of a CROF device, one plate is X-Plate andthe other plate is a planar thin film, wherein the thickness of at leastone of the plates is in a range of from 10 um to 250 um; wherein thespacers are fixed on the X-Plate, and are an array of square orrectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-100um, and wherein the plates and the spacers can have the same materialsor different materials and are made of PMMA (polymethyl methacrylate),PS (polystyrene), or a material of similar mechanical properties as PMMAor PS.

The “similar” in above paragraphs means that the difference inmechanical properties within 60%.

Guard Ring.

Some embodiments have a guard ring to prevent sample flow out of theplate surface. Some embodiments of the guard ring is an enclosed wallaround the sample area. The wall has a height equal to the spacer heightor different from the spacer height. The wall ca be a significantdistance away from the sample measurement area.

The movable plates in a CROF process may include and/or may be coupledto a hinge, a stage, or some other positioning system that is configuredto transition the plates between an open configuration and a closedconfiguration. Movable plates may be coupled together with one or morejoints in a manner that leaves an opening to access the space betweenthe plates (e.g., to insert and/or remove sample), provided that atleast one of the joints and/or at least one of the plates is flexibleenough to achieve the described open and closed configurations. Amembrane pump is not considered to be a movable plate(s).

3. Analytes, Entity, Binding Site, Storage Site

In present invention, the entity include, but not limited to, one of aprotein, an amino acid, a nucleic acid, a lipid, a carbohydrate, ametabolite, a cell, or a nanoparticle.

In some embodiments, the binding site includes a binding partnerconfigured to bind to the respective entity.

In some embodiments, the binding site includes an entity bound to thebinding site. In some embodiments, the placing the sample includesplacing the sample within the binding site.

In some embodiments, the reagent includes at least one of a protein, anamino acid, a nucleic acid, a lipid, a carbohydrate, and a metabolite.

In certain embodiments, the storage site includes dried reagent.

In some embodiments, the storage site includes reagent configured to bereleased from the storage site upon contact with the sample.

In some embodiments, the first storage site and the second storage siteare in a common storage site.

In some embodiments, the transfer media is a sample. In someembodiments, the transfer media is a liquid, wherein the reagent or theentity can be dissolved and diffuse in the liquid.

In some embodiments, a plate has multiple storage sites. In anotherembodiment, one storage site has multiple reagent.

Different Release Time.

In some embodiments, a plate has multiple storage sites on differentlocations of the plate or one storage site stores multiple reagent, andupon in touch with the sample by the storage sites, the reagents arereleased but released at different time for different reagents on thesame storage site or reagents on different storage sites.

In some embodiments, the first reagent is configured to be released fromthe first storage site upon contact with the sample in a first averagerelease time and the second reagent is configured to be released fromthe second storage site upon contact with the sample in a second averagerelease time, and wherein the first average release time is less thanthe second average release time.

In some embodiments, the first reagent is configured to be released fromthe first storage site upon contact with the sample and wherein thesecond reagent is a bound reagent.

In some embodiments, the depositing includes binding at least one of thereagents to the respective plate.

In some embodiments, the contacting includes releasing at least one ofthe reagents from the respective plate.

In some embodiments, the depositing includes depositing a first reagentand a second reagent, and wherein the contacting includes releasing thefirst reagent before the second reagent.

In some embodiments, at least one of the plates comprises a storage sitethat includes a reagent that is to be added to the relevant volume ofthe sample.

In some embodiments, wherein the reagent includes at least one of aprotein, an amino acid, a nucleic acid, a lipid, a carbohydrate, and ametabolite.

In some embodiments, the storage site includes dried reagent.

In some embodiments, the storage site includes reagent configured to bereleased from the storage site upon contact with the sample.

In some embodiments, the storage site is a first storage site and thereagent is a first reagent, wherein the device includes a second storagesite including a second reagent that is to be added into the relevantvolume of the sample, wherein the second storage site is on one of theplates.

In some embodiments, the first storage site and the second storage siteare in a common storage site.

In some embodiments, the first reagent is configured to be released fromthe first storage site upon contact with the sample in a first averagerelease time and the second reagent is configured to be released fromthe second storage site upon contact with the sample in a second averagerelease time, and wherein the first average release time is less thanthe second average release time.

In some embodiments, at least one of the reagents is dried on therespective plate.

In some embodiments of a kit, at least one of the reagents is bound tothe respective plate.

In some embodiments of a kit, at least one of the reagents is configuredto be released from the respective plate upon contact with the sample.

In some embodiments of a kit, a first reagent is on one or both of theplates and a second reagent is on one or both of the plates, wherein thefirst reagent is configured to be released from the respective plateupon contact with the sample in a first average release time and thesecond reagent is configured to be released from the respective plateupon contact with the sample in a second average release time, andwherein the first average release time is less than the second averagerelease time.

In some embodiments of the devices, the storage site is a first storagesite and the reagent is a first reagent, wherein the device includes asecond storage site including a second reagent that is to be added intothe relevant volume of the sample, wherein the second storage site is onone of the plates.

4. Locally Binding or Mixing in a Portion of a Sample (P)

In some applications, it is desirable to have a binding site to capture(i.e. bind) the analytes only in a portion of a sample, not in theentire sample. It is also desirable in some cases that a reagent isadded (i.e. mixed) into a port of a sample, not the entire sample. It isoften desirable that there is no fluidic separation between the portionof the sample and the rest of the sample. Such requirements arepreferable or necessary in certain multiplexed detections.

The present invention offers a solution to the above requirements byusing a CROF method and device to reshape a sample into a ultra-thinfilm of a thickness, that is smaller than the lateral dimension of theportion of the sample, wherein only an analyte inside that portion ofthe sample will be captured, or only the portion of the sample will bemixed with a reagent. The working principle for such approach is thatwhen the thickness of the sample is smaller than the lateral dimensionof the portion of the sample, a capture of an analyte by a surface or amixing of reagent placed on a surface can be primarily limited by thediffusion of the analytes and the reagent in the thickness direction,where the diffusion in the lateral diffusion is relativelyinsignificant. For example, if a sample is reshaped in to a thin film of5 um thick, if the portion of the sample that an analyte should becaptured or a reagent should be mixed has a lateral dimension of 5 mm by5 mm, and if the diffusion time of analyte or reagent across the 5 um is10 sec, then the lateral diffusion of the analyte or the reagent acrossthe 5 mm distance is 1,000,000 sec (since the diffusion time isproportional to the square of the diffusion distance). This means thatby selecting a proper ratio of the lateral dimension of the interestedportion of the sample to the sample thickness, in certain time interval,the analytes captured primarily come from the sample portion interested,or the regent is mixed primarily into the portion of the sample ofinterest.

4.1 Locally Binding of Entity in a Portion of a Sample to a Surface (P:Volume to Surface)

P1. A method for locally bind target entities in a relevant volume of asample to a binding site on a surface, comprising:

-   -   (i) perform the steps of (a) to (d) in the method of paragraph        X1, wherein the sample thickness at the closed configuration is        significantly less than the average linear dimension of the        binding site; and wherein the relevant volume is the volume of        the sample that sits on the binding site when the plates are in        the closed configuration;    -   (ii) after (i) and while the plates are in the closed        configuration, either.        -   (1) incubating the sample for a relevant time length and            then stopping the incubation; or        -   (2) incubating the sample for a time that is equal or longer            than the minimum of a relevant time length, and then            assessing, within a time period that is equal or less than            the maximum of the relevant length of time, the binding of            target entity to in the binding site;    -   wherein the relevant time length is:        -   i. equal to or longer than the time that it takes for the            target entity to diffuse across the thickness of the uniform            thickness layer at the closed configuration; and        -   ii. significantly shorter than the time that it takes the            target entity to laterally diffuse across the minimum            lateral dimension of the binding site;    -   wherein at the end of the incubation in (1) or during the        assessing in (2), the majority of the target entity bound to the        binding site is from a relevant volume of the sample;    -   wherein the incubation allows the target entity to bind to the        binding site, and wherein the relevant volume is a portion of        the sample that is above the binding site at the closed        configuration.

The method of paragraph P2, wherein the term “the thickness of arelevant volume of the sample is significantly less than the minimumaverage dimension of the binding site” means that the ratio of theminimum average dimension of the binding site to the sample thickness(termed “length to thickness ratio”) is at least 3, at least 5, at least10, at least 20, at least 50, at least 100, at least 500, at least1,000, at least 10,000, at least 100,000, or any range between thevalues. In preferred embodiments, the length to thickness ratio is atleast 3, at least 5, at least 10, at least 20, at least 50, at least100, at least 500, or any range between the values.

The method of paragraph P2, wherein the term “significantly shorter thanthe time that it takes the target entity to laterally diffuse across theminimum lateral dimension of the binding site” means that the ratio ofthe time for diffusing across the minimum lateral dimension of thebinding site to the time for diffusion across the sample thickness(termed “length to thickness diffusion time ratio”) is at least 3, atleast 10, at least 50, at least 10, at least 100, at least 1,000, atleast 10,000, at least 100,000, at least 100,000, or any range betweenthe values. In preferred embodiments, the length to thickness diffusiontime ratio is at least 3, at least 10, at least 50, at least 10, atleast 100, at least 1,000, at least 10,000, or any range between thevalues.

P2. A device for locally binding entity in a relevant volume of a sampleto a binding site on surface, comprising:

-   -   a first plate and a second plate, that are movable relative to        each other into different configurations,    -   wherein the first plate has, on its surface, a binding site that        has an area smaller than that of the plate and is configured to        bind target entity in a sample, wherein the target entity are        capable of diffusing in the sample, and wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers, the binding site, and at least a        portion of the sample are between the plates, the sample        contacts at least a part of the binding site, the thickness of a        relevant volume of the sample is regulated by the plates and the        spacers, is thinner than the maximum thickness of the sample        when the plates are in the open configuration, wherein the        relevant volume is the volume of the sample that sits on the        binding site;    -   wherein the spacer height is selected to regulate the thickness        of the relevant volume at the closed configuration to be at        least 3 times less than the average linear dimension of the        binding site.

The regulation of the thickness of the relevant volume to 3 times lessthan the average linear dimension of the binding site makes thediffusion time of the entity across the sample thickness is 9 times lessthan that across a distance equal to the average linear dimension of thebinding site. Such thickness regulation makes it possible to select anincubation time, such that the incubation results in (i) a significantnumber of target entity in the relevant volume are bound to the bindingsite and (ii) a significant number of the target entity bound to thebinding site are from the relevant volume of the sample, and wherein theincubation is a process to allow the target entity to bind to thebinding site.

For example, if the incubation time is set to be the time that equals tothe diffusion time of the entity across the thickness of the relevantvolume of the sample, then after the incubation, most of the entityinside the relevant volume are already reached the binding site andbeing bound according to the rate equation, while the entity originally(i.e. before the incubation) outside of the relevant volume can onlydiffuse into the peripheral of the relevant volume (relative smallvolume) and such volume becomes less significant, as the ratio of theaverage linear dimension of the binding site to the relevant volumethickness gets larger.

4.2 Locally Binding Entity Stored on a Plate Surface to a Binding-Siteon Other Plate Surface (Surface to Surface)

P3. A method for locally binding entity stored on a storage site of oneplate to a binding site on another plate, comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein a        surface of first plate has a binding site; and a surface of the        second plate has a storage site that comprises entity to be        bound to the binding site; wherein the area of the binding site        and the area of the reagent site is less than that of respective        plates; and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   (b) obtaining a transfer medium, wherein the entity are capable        of being dissolving into the transfer medium and diffusing in        the transfer medium;    -   (c) depositing, when the plates are configured in an open        configuration, the transfer medium on one or both of the plates;        wherein the open configuration is a configuration in which the        two plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the transfer medium by bringing the        plates into a closed configuration, wherein, in the closed        configuration: the plates are facing each other, the spacers,        the binding site, the storage site and at least a portion of the        transfer medium are between the plates; at least a portion of        the storage site is directly facing the binding site with a        portion of the transfer medium between them, and the thickness        of a relevant volume of the transfer medium is regulated by the        plates and the spacers, is thinner than the maximum thickness of        the sample when the plates are in the open configuration, and is        significantly less than the average linear dimension of the        relevant volume in the plate surface direction; and    -   (e) after (d) and while the plates are in the closed        configuration, incubating for a time and stopping the        incubation, wherein the incubation time is selected in such that        results in a significant number of the entity bound to the        binding site are from the storage site, wherein the relevant        volume is the volume of the transfer medium that sits on the        binding site and the incubation is a process to allow the entity        to bind to the binding site.

The term of “at least a port of the storage site is directly facing thebinding site” means that the shortest distance from a point in theportion to the binding site is the same as the thickness of the relevantvolume at the closed configuration of the plates.

P4. A device for binding entity stored on a storage site of one plate toa relevant binding site on another plate, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations, wherein a surface of        first plate has a binding site; and a surface of the second        plate has a storage site that contains entity to be bound to the        binding site; wherein the area of the binding site and the area        of the storage site is less than that of respective plates; and        wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and a transfer medium is deposited on one or        both of the plates, wherein the entity on the storage site are        capable of being dissolving into the transfer medium and        diffusing in the transfer medium,    -   wherein another of the configuration is a closed configuration,        which is configured after the transfer medium deposition in the        open configuration; and in the closed configuration: the plates        are facing each other, the spacers, the binding site, the        storage site and at least a portion of the transfer medium are        between the plates; at least a portion of the storage site is        directly facing the binding site with a portion of the transfer        medium between them, and the thickness of a relevant volume of        the transfer medium is regulated by the plates and the spacers,        and is thinner than the maximum thickness of the sample when the        plates are in the open configuration;    -   wherein the relevant volume is the volume of the transfer medium        that sits on the storage site when the plates are in closed        configuration; and    -   wherein the spacer height is selected to regulate the thickness        of the relevant volume at the closed configuration to be at        least 3 times less than the average linear dimension of the        binding site.    -   wherein at least one of the spacers is inside the sample contact        area;    -   and the spacers that have a predetermined inter-spacer distance        and height.        4.3 A Method for locally binding entity on multiple storage        sites of one plate to multiple corresponding binding sites on        another plate

P5. A method for locally binding entity stored on multiple storage sitesof one plate to multiple corresponding binding sites on another plate,comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations; wherein a        surface of first plate has multiple binding sites, and a surface        of the second plate has multiple corresponding storage sites;        wherein each corresponding storage site is located in a location        on the second plate that is corresponding to the location of a        binding site, so that when the two plates are placed        face-to-face, each binding site overlaps only one storage site;        and wherein one or both of the plates comprise spacers and each        of the spacers is fixed with its respective plate and has a        predetermined height;    -   (b) obtaining a transfer medium, wherein the entity on the        storage sites are capable of being dissolving into the transfer        medium and diffusing in the transfer medium;    -   (c) depositing, when the plates are configured in an open        configuration, the transfer medium on one or both of the plates;        wherein the open configuration is a configuration in which the        two plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the transfer medium by bringing the        plates into a closed configuration, wherein, in the closed        configuration: the two plates are facing each other, the        spacers, the binding sites, the storage sites and at least a        portion of the transfer medium are between the plates, each        binding site directly faces only one corresponding storage site,        the transfer medium contacts at least a part of each of the        binding sites and a part of each of the storage sites, the        thickness of a relevant volume of the transfer medium is        regulated by the plates and the spacers, is thinner than the        maximum thickness of the transfer medium when the plates are in        the open configuration, and is significantly less than the        average linear dimension of the binding sites; and    -   (e) after (d) and while the plates are in the closed        configuration, incubating for a time and stopping the        incubation, wherein the incubation time is selected in such that        results in a significant number of the entity bound to each        binding site are from a corresponding storage site, wherein the        relevant volume is the volume of the transfer medium that sits        on the binding sites, and the incubation is a process to allow        the entity to be bound to the binding site.

In some embodiments the spacing is limited to the binding sample area.

In some embodiments of the method P5, the transfer medium is a samplewith target analyte, the binding site comprises capture agent, and theentity in the storage site is detection agent, wherein the targetanalyte binds the capture agent and the detection agent to form acapture agent-analyte-detection agent sandwich. The method P5 simplifyan assay steps and can reduce the assay time by using smaller spacerheight to have a thinner sample thickness and shorter vertical diffusiontime for both analytes and detection agents for a shorter saturationassay time.

P6. A device for locally binding entity stored on multiple storage sitesof one plate to multiple corresponding binding sites on another plate,comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein a surface of first plate has multiple binding sites, and        a surface of the second plate has multiple corresponding storage        sites; wherein each corresponding storage site is located in a        location on the second plate that is corresponding to the        location of a binding site, so that when the two plates are        placed face-to-face, each binding site overlaps only one storage        site; and wherein one or both of the plates comprise spacers and        each of the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and a transfer medium is deposited on one or        both of the plates, wherein the entity on the storage site are        capable of being dissolving into the transfer medium and        diffusing in the transfer medium,    -   wherein another of the configuration is a closed configuration,        which is configured after the transfer medium deposition in the        open configuration; and in the closed configuration: the two        plates are facing each other, the spacers, the binding sites,        the storage sites and at least a portion of the transfer medium        are between the plates, each binding site directly faces only        one corresponding storage site, the transfer medium contacts at        least a part of each of the binding sites and a part of each of        the storage sites, the thickness of a relevant volume of the        transfer medium is regulated by the plates and the spacers, and        is thinner than the maximum thickness of the transfer medium        when the plates are in the open configuration;    -   wherein the relevant volume is the volume of the transfer medium        that sits on the storage site when the plates are in closed        configuration; and    -   wherein the predetermined spacer height is selected to regulate        the thickness of the relevant volume at the closed configuration        to be significantly less than the average linear dimension of        the binding sites.

4.4 Locally Adding Reagent Stored on a Surface to a Portion of a Sample(Surface to Volume)

P7. A method for locally adding a reagent into a relevant volume of asample, comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a storage site that        contains reagents to be added into a relevant volume of a        sample, the reagents are capable of being dissolving into the        sample and diffusing in the sample, and the area of the storage        site is less than that of the plate; and wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   (b) obtaining the sample;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration; wherein, in the closed configuration:        the plates are facing each other; the spacers, the storage site,        and at least a portion of the sample are between the plates; the        sample contacts at least a portion of the storage site and        contacts the plates over an area that is larger than that of the        storage site; the thickness of a relevant volume of the sample        is regulated by the plates and the spacers, is thinner than the        maximum thickness of the sample when the plates are in the open        configuration, and is significantly less than the average linear        dimension of the relevant volume in the plate surface direction;        and    -   (e) after (d) and while the plates are in the closed        configuration, incubating for a time and stopping the        incubation, wherein the incubation time is selected in such that        results in (i) a significant number of the reagents dissolved in        the sample are contained in the relevant volume of the sample        and (ii) the reagents are in the significant part of the        relevant volume, and wherein the relevant volume is the volume        of the sample that sits on the storage site when the plates are        in closed configuration, and the incubation is a process to        allow the reagent to dissolve and diffuse in the sample.

P8. A device for locally adding a reagent stored on a plate surface intoa relevant volume of a sample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations,    -   wherein the first plate has, on its surface, a storage site that        contains reagents to be added into a relevant volume of a        sample, the reagents are capable of being dissolving into the        sample and diffusing in the sample; and wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers, the storage site, and at least a        portion of the sample are between the plates, the sample        contacts at least a portion of the storage site and at least a        port of plate surface outside the storage site, the thickness of        a relevant volume of the sample is regulated by the plates and        the spacers, is thinner than the maximum thickness of the sample        when the plates are in the open configuration, and wherein the        relevant volume is the volume of the sample that sits on the        storage site when the plates are in closed configuration;    -   wherein the spacer height is selected to regulate the thickness        of the relevant volume at the closed configuration of the plates        to be at least 3 times less than the average linear dimension of        the relevant volume in the plate surface direction.

5. Formation of Capture-Analyte-Detection Sandwich on a Binding Site (W)

One aspect of the present invention is to form acapture-analyte-detection sandwich on a binding site on a solid surfacein a single step by using a CROF process and by putting the binding siteon one plate and a storage site which stores the detection agent on thecorresponding location of the other plate.

5.1 Forming Capture-Analyte-Detection Sandwich on a Binding Site in aSingle Step of Incubation (General) (W)

W1. A method for forming a capture-analyte-detection sandwich on abinding site of a plate, comprising:

-   -   (a) obtaining a sample that contains a target analyte, wherein        the target analyte is capable of diffusion in the sample;    -   (b) obtaining capture agents and obtaining detection agents,        wherein the capture agents and the detection agents (are capable        to) bind to the target analyte to form a capture agent-target        analyte-detection agent sandwich;    -   (c) obtaining a first plate and a second plate that are movable        relative to each other into different configurations; wherein        the first plates has a binding site that has the capture agents        being immobilized on the site, and the second plate has a        storage site that stores the detection agents; wherein when the        storage site is in contact with the sample, the detection agents        are capable to be dissolved into the sample and diffuse in the        sample; and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   (d) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (e) after (d), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers, and is thinner than the sample thickness when the        plates are in the open configuration, and the sample is in        contact with the binding site and the storage site; and    -   (f) after (e), while the plates are in the closed configuration,        incubating for a time to allow a formation of capture        agent-target analyte-detection agent sandwich;    -   wherein the relevant volume is at least a portion or an entire        volume of the sample.

W2. A device for forming a capture-analyte-detection sandwich on abinding site of a plate, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plates has a binding site that has capture        agents being immobilized on the site, and the second plate has a        storage site that stores detection agents; wherein the capture        agents and the detection agents (are capable to) bind to a        target analyte in a sample to form a capture agent-target        analyte-detection agent sandwich; wherein when the storage site        is in contact with the sample, the detection agents are capable        to be dissolved into the sample and diffuse in the sample; and        wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and a relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than the sample thickness when the plates are in        the open configuration, and the sample is in contact with the        binding site and the storage site; and    -   wherein the relevant volume is at least a portion or an entire        volume of the sample.        5.2 Forming Capture-Analyte-Detection Sandwich on a Binding Site        in a Single Step Incubation Using the Analyte that is from a        Portion of the Sample (i.e. Locally).

W3. A method for forming a capture-analyte-detection sandwich on abinding site of a plate using the analytes that are from a portion ofthe sample, comprising:

-   -   (a) obtaining a sample that contains a target analyte, wherein        the target analyte is capable of diffusion in the sample;    -   (b) obtaining capture agents and obtaining detection agents,        wherein the capture agents and the detection agents are capable        to bind to the target analyte to form a capture agent-target        analyte-detection agent sandwich;    -   (c) obtaining a first plate and a second plate that are movable        relative to each other into different configurations; wherein        the first plates has a binding site that has the capture agents        being immobilized on the site, and the second plate has a        storage site that stores the detection agents, which, when the        reagent a storage site is in contact with the sample, are        capable to be dissolved into the sample and diffuse in the        sample; and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   (d) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (e) after (d), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers, the binding site,        and the storage site are between the plates, the binding site        and the storage site are in contact with a relevant volume of        the sample, and the thickness of the relevant volume of the        sample is regulated by the plates and the spacers and is thinner        than the sample thickness when the plates are in the open        configuration; and is significantly less than the average linear        dimension of the binding site; and    -   (f) after (e) and while the plates are in the closed        configuration, incubating for a time and stopping the        incubation, wherein the incubation time is selected in such that        results in a significant number of the capture-analyte-detection        sandwich formed at the binding site contain the analytes that        come from the relevant volume of the sample, wherein the        relevant volume is the volume of the sample that sits on the        binding site, and the incubation is a process to allow a        formation of a capture-analyte-detection sandwich.

In some embodiments the ratio of the spacing to the site dimension maybe less than ⅕.

W4. A device for forming a capture-analyte-detection sandwich on abinding site of a plate with the analytes that are from a portion of thesample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plates has a binding site that has capture        agents being immobilized on the site, and the second plate has a        storage site that stores detection agents; wherein the capture        agents and the detection agents (are capable to) bind to a        target analyte in a sample to form a capture agent-target        analyte-detection agent sandwich; wherein when the storage site        is in contact with the sample, the detection agents are capable        to be dissolved into the sample and diffuse in the sample; and        wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers, the binding site, and the        storage site are between the plates, the binding site and the        storage site are in contact with a relevant volume of the        sample, and the thickness of the relevant volume of the sample        is regulated by the plates and the spacers and is thinner than        the sample thickness when the plates are in the open        configuration; and wherein the relevant volume is the volume of        the sample that sits on the binding site; and    -   wherein the spacer height is selected to regulate the thickness        of the relevant volume at the closed configuration to be        significantly less than the average linear dimension of the        binding site.

5.3 A Method for Reducing the Time of Forming Capture-Analyte-DetectionSandwich on a Binding Site by Reducing the Diffusion Distance (W, X).

W5. A method for reducing the time of forming acapture-analyte-detection sandwich on a binding site of a plate,comprising:

-   -   (a) obtaining a sample that contains a target analyte, wherein        the target analyte is capable of diffusion in the sample;    -   (b) obtaining capture agents and obtaining detection agents,        wherein the capture agents and the detection agents are capable        to bind to the target analyte to form a capture agent-target        analyte-detection agent sandwich;    -   (c) obtaining a first plate and a second plate that are movable        relative to each other into different configurations; wherein        the first plates has a binding site that has the capture agents        being immobilized on the site, and the second plate has a        storage site that stores the detection agents, which, when the        reagent a storage site is in contact with the sample, are        capable to be dissolved into the sample and diffuse in the        sample; and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   (d) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (e) after (d), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers, the binding site,        and the storage site are between the plates, the binding site        overlaps the storage site, the binding site and the storage site        are in contact with a relevant volume of the sample, and the        thickness of the relevant volume of the sample is regulated by        the plates and the spacers and is thinner than the sample        thickness when the plates are in the open configuration; and        thereby the reduced thickness of the sample reduces the time for        the analytes and the detection agents diffusing vertically        across the thickness of the sample, wherein the relevant volume        is at least a portion of an entire volume of the sample.        wherein the time period to allow the target entity in the        relevant volume to bind to the binding site is shorter than that        without the closed configuration.    -   the method may further comprise a wash step to remove the sample        between the plates, and the wash step is performed when the        plates are in either a closed configuration or an open        configuration.    -   The methods further comprise a read step that reads the signal        from the capture-analyte-detection sandwich immobilized on the        binding site. The read is performed either after a wash or        without any wash.        The method may further be multiplexed, as described above or        below.

W6. A device for reducing the time of forming acapture-analyte-detection sandwich on a binding site of a plate,comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plates has a binding site that has capture        agents being immobilized on the site, and the second plate has a        storage site that stores detection agents; wherein the capture        agents and the detection agents (are capable to) bind to a        target analyte in a sample to form a capture agent-target        analyte-detection agent sandwich; wherein when the storage site        is in contact with the sample, the detection agents are capable        to be dissolved into the sample and diffuse in the sample; and        wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers, the binding site, and the        storage site are between the plates, the binding site overlaps        the storage site, the binding site and the storage site are in        contact with a relevant volume of the sample, and the thickness        of the relevant volume of the sample is regulated by the plates        and the spacers and is thinner than the sample thickness when        the plates are in the open configuration; and thereby the        reduced thickness of the sample reduces the time for the        analytes and the detection agents diffusing vertically across        the thickness of the sample, wherein the relevant volume is at        least a portion of an entire volume of the sample.

In these embodiments, the method may comprise attaching a capture agenta plate, wherein the attaching is done via a chemical reaction of thecapture agent with a reactive group on the plate. The other plate maycontain a patch of a dried detection reagent at a location such that,after the plates are closed, the affixed capture agent and the patch ofdetection reagent are facing each other. Next, the method may comprisecontacting a sample containing a target-analyte with the device andclosing the plates, as described above. The detection reagent dissolvesand diffuses into the sample. Since the target analyte is in solution,the target analyte will be bound by the capture agent and immobilized tothe surface of one of the plates. The detection agent can bind to thetarget analyte before or after it is bound to the capture agent. In somecases, the method may comprises removing any target-analytes that arenot bound to the capture agent, or any unbound detection reagent (e.g.,by washing the surface of a plate in binding buffer); The detectionagent may be conjugated with an optical detectable label, therebyproviding a way to detect the target analyte. After optionally removingthe detection agent that are not bound to the target-analyte, the systemcan be read, e.g., using a reading system, to read a light signal (e.g.,light at a wavelength that is in the range of 300 nm to 1200 nm) fromdetection agent that is bound to the plate. Further, as mentioned above,the detection agent may be labeled directly (in which case the detectionagent may be strongly linked to a light-emitting label prior todeposition onto one of the plates), or labeled indirectly (i.e., bybinding the detection agent to a second capture agent, e.g., a secondaryantibody that is labeled or a labeled nucleic acid, that specificallybinds to the detection agentt and that is linked to a light-emittinglabel). In some embodiments, the method may comprise a blocking agent,thereby preventing non-specific binding of the capture agents tonon-target analytes. Suitable conditions for the specific binding oftarget analytes to other agents, include proper temperature, time,solution pH level, ambient light level, humidity, chemical reagentconcentration, antigen-antibody ratio, etc., are all well known orreadily derivable from the present disclosure. General methods formethods for molecular interactions between capture agents and theirbinding partners (including analytes) are well known in the art (see,e.g., Harlow et al., Antibodies: A Laboratory Manual, First Edition(1988) Cold spring Harbor, N.Y.; Ausubel, et al, Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons, 1995). The methods describedabove and below are exemplary; the methods herein are not the only waysof performing an assay.

In certain embodiments, a nucleic acid capture agent can be used tocapture a protein analyte (e.g., a DNA or RNA binding protein). Inalternative embodiments, the protein capture agent (e.g., a DNA or RNAbinding protein) can be used to capture a nucleic acid analyte.

The sample may be a clinical sample derived from cells, tissues, orbodily fluids. Bodily fluids of interest include but are not limited to,amniotic fluid, aqueous humour, vitreous humour, blood (e.g., wholeblood, fractionated blood, plasma, serum, etc.), breast milk,cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph,perilymph, feces, gastric acid, gastric juice, lymph, mucus (includingnasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleuralfluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat,synovial fluid, tears, vomit, urine and exhaled condensate.

In one embodiment of this assay, a plate is contacted with a samplecontaining a target analyte (e.g., a target protein) and the plates areclosed. The sample contains, or is amended to contain, all necessaryreagents (e.g., salts and the like) conditions suitable for specificbinding. The capture agents (e.g., antibodies) and detection agentspecifically bind to a target analyte in the sample, thereby leading toa patch of labeled analyte that can be detected.

As in any embodiment, the amount of target analyte in the sample can bemeasured to provide a qualitative or quantitative measure of the amountof target analyte in the sample. In some embodiments, the magnitude ofthe signal provides a quantitative determination of the amount of targetanalyte in the sample. In some cases, the evaluation may be compared toa standard curve (e.g., of a second analyte or a spiked-in analyte) thatmay in certain cases be at a known concentration. This comparison may befacilitated by depositing capture agents at different densities (e.g.,different concentrations) and reading the signal from each patch ofcapture agent.

6 Binding and Adding Using Samples and Reagent with Small Volume (V)

It is highly desirable, in many applications, to use as small volume ofa sample or reagent as possible. However, in microfluidic channeldevices (the most popular approach today for using small samples), asignificant volume of the sample is wasted in flowing from an inlet to atesting (detection) region of the device, resulting a need to a samplevolume larger than the volume in the testing location. One aspect of thepresent invention is to significantly reduce the volume of the sample orreagent used in a testing, by depositing a tiny volume of a sample or areagent on a plate and then reshaping the volume into a thin film with asmaller thickness but larger area than before. Such reshaping alsoallows faster reaction.

6-1 Binding Target Entity in a Small Volume Sample on a Surface BindingSite by Spreading the Sample.

V1. A method for binding target entity in a sample to a binding site,comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a binding site, and wherein        one or both of the plates comprise spacers and each of the        spacers is fixed with its respective plate and has a        predetermined height;    -   (b) obtaining a sample that contains a target entity to be bound        to the binding site;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein,        in the open configuration: the two plates are partially or        completely separated apart, the spacing between the plates is        not regulated by the spacers, and the sample, as deposited,        covers either no area or a partial area of the binding site;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration; wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the sample contacts        more area of the binding site than that when the plates are in        the open configuration, and the thickness of the relevant volume        of the sample on the binding site is regulated by the plates and        the spacers, wherein the relevant volume is a portion or an        entire volume of the sample.

V2. A device for binding target entity in a sample to a surface bindingsite, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plate has, on its surface, a binding site that        binds target entity in a sample, and wherein the binding site        has an area larger than the contact area of the sample when the        sample is deposited on only one of the plates and without        contacting the other plate;    -   wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates and covers, as deposited, either no area or a partial        area of the binding site;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the sample are between the        plates, the sample contacts more area of the binding site than        that when the plates are in the open configuration, and the        thickness of the sample on the binding site is regulated by the        plates and the spacers.        6-2 Adding Reagents into a Small Volume Sample by Spreading the        Sample

V3. A method for binding target entity in a sample to a binding site,comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a storage site that        contains the reagents to be added into the sample, and wherein        one or both of the plates comprise spacers and each of the        spacers is fixed with its respective plate and has a        predetermined height;    -   (b) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein,        in the open configuration: the two plates are partially or        completely separated apart, the spacing between the plates is        not regulated by the spacers, and the sample, as deposited,        contacts either no area or a partial area of the storage site;    -   (c) after (b), spreading the sample by bringing the plates into        a closed configuration; wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the sample contacts        more area of the storage site than that when the plates are in        the open configuration, and the thickness of the relevant volume        of the sample is regulated by the spacer; and wherein the        relevant volume is a portion of the sample that site on the        storage site.

V4. A device for binding target entity in a sample to a binding site,comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations,    -   wherein the first plate has, on its surface, a storage site that        contains reagents and the reagents are to be added into the        sample, and wherein one or both of the plates comprise spacers        and each of the spacers is fixed with its respective plate and        has a predetermined height;    -   wherein one or both of the plates comprise spacers and each of        the spacers is fixed with its respective plate and has a        predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and a relevant volume of the        sample are between the plates, the sample contacts more areas of        the storage site than that when the plates are in the open        configuration, and the thickness of the relevant volume of the        sample is regulated by the spacer; and wherein the relevant        volume is a portion of the sample that site on the storage site.

In the methods of paragraph V1 and V2 and the devices of V3 and V4, insome cases, even a sample is deposited in the binding site area or thestorage area, due to the small volume of the sample and a wettingproperty of the surface, the contact area of as-deposited sample with aplate will be less than the area of the binding site or the storagesite. Hence, a spreading, particular precisely spreading is needed.

Drops of a sample can be multiple drops, and in the closedconfiguration, the drops merged into a film with a thickness less thanthe maximum thickness.

In present invention, in the method in paragraph V1 to V7 and thedevices in paragraph of V2 to V8, the volume of the sample that isdeposited on the plate or the plates (“sample volume”) is at most 0.001pL (pico liter), at most 0.01 pL, at most 0.1 pL, at most 1 pL, at most10 pL, at most 100 pL, at most 1 nL (nano liter), at most 10 nL, at most100 nL, at most 1 uL (micro liter), at most 10 uL, at most 100 uL, atmost 1 mL (milliliter), at most 10 mL, or a range of any two of thesevalues.

7 Uniform Binding or Adding Reagents Using Uniform Sample Thickness(UAB)

For assays and chemical reactions, it is advantageous to make a thinsample thickness uniform over a significant area. The examples includebinging of entity of sample to a surface binding site, adding reagentsinto a sample, quantification a relevant volume of the sample,quantification of analytes, and others. For the methods that use twoplates to reduce and regulate a thickness of a relevant volume (aportion or an entire volume) of a sample, it is essential to be precise,uniform and easy-to-use.

One aspect of the present invention is to improve the precision,uniformity, or easy-to-use of the methods and/or devices that regulate athickness of a relevant volume of a sample by compressing the samplewith two plates.

7.1 A Method for Uniformly Binding an Entity in a Sample into a BindingSite of a Plate

UAB1. A method for uniformly binding an entity in a sample into abinding site of a plate, comprising:

-   -   (a) obtaining a sample that contains target entity which are        capable of diffusing in the sample;    -   (b) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a binding site that is        configured to bind the target entity, wherein one or both of the        plates comprise spacers and each of the spacers is fixed with        its respective plate and has a predetermined height    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the binding site is        in contact with the relevant volume, the thickness of the        relevant volume of the sample is regulated by the plates and the        spacers and is, compared to the plates are in the open        configuration, thinner than the maximum thickness of the sample        and more uniform over the binding site;        -   wherein the spacers and the plate are configured to make the            regulated thickness of the relevant volume at the plate            closed configuration more uniform than that in the plate            open configuration; and wherein the relevant volume is a            portion or an entire volume of the sample.        -   It further has a storage site on the plate opposite to the            binding site for forming a uniform sandwich.

UAB2. A device for uniformly binding an entity in a sample into abinding site on a plate, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plate has, on its surface, a binding site that        is configured to bind the target entity, wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the binding site is in contact        with the relevant volume, the thickness of the relevant volume        of the sample is regulated by the plates and the spacers and is,        compared to the plates are in the open configuration, thinner        than the maximum thickness of the sample and more uniform over        the binding site;    -   wherein the spacers and the plates are configured to make the        regulated thickness of the relevant volume at the plate closed        configuration more uniform than that in the plate open        configuration; and wherein the relevant volume is a portion or        an entire volume of the sample.        7.2 A Method for Uniformly Adding a Regent on a Plate into a        Sample

UAB3. A method for uniformly adding a reagent into a relevant volume ofa sample, comprising:

-   -   (a) obtaining a first plate and a second plate that are movable        relative to each other into different configurations, wherein        the first plate has, on its surface, a storage site that        contains reagents to be added into a relevant volume of a        sample, the reagents are capable of being dissolving into the        sample and diffusing in the sample; and wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   (b) obtaining the sample;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the storage site is        in contact with the relevant volume, and the thickness of the        relevant volume of the sample is regulated by the plates and the        spacers and is thinner than the maximum thickness of the sample        when the plates are in the open configuration;        -   wherein the spacers and plates are configured to make the            thickness of the relevant volume of the sample more uniform            over the area of the relevant volume at the plate closed            configuration than that at the plate open configuration; and            wherein the relevant volume is a portion or an entire volume            of the sample.

UAB4. A device for uniformly adding a reagent into a relevant volume ofa sample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein the first plate has, on its surface, a storage site that        contains reagents to be added into a relevant volume of a        sample, the reagents are capable of being dissolving into the        sample and diffusing in the sample; and wherein one or both of        the plates comprise spacers and each of the spacers is fixed        with its respective plate and has a predetermined height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the storage site is in contact        with the relevant volume, and the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than the maximum thickness of the sample when the        plates are in the open configuration;    -   wherein the spacers and plates are configured to make the        thickness of the relevant volume of the sample more uniform over        the area of the relevant volume at the plate closed        configuration than that at the plate open configuration; and        wherein the relevant volume is a portion or an entire volume of        the sample.

7.3 A Method for Uniformly Forming a Capture-Analyte-Detection Sandwich

UAB5. A method for uniformly a capture-analyte-detection sandwich on abinding site of a plate, comprising:

-   -   (a) obtaining a sample that contains a target analyte;    -   (b) obtaining capture agents and obtaining detection agents,        wherein the capture agents and the detection agents (are capable        to) bind to the target analyte to form a capture agent-target        analyte-detection agent sandwich;    -   (c) obtaining a first plate and a second plate that are movable        relative to each other into different configurations; wherein        the first plates has a binding site that has the capture agents        being immobilized on the site, and the second plate has a        storage site that stores the detection agents, which, when the        storage site is in contact with the sample, are capable to be        dissolved into the sample and diffuse in the sample; and wherein        one or both of the plates comprise spacers and each of the        spacers is fixed with its respective plate and has a        predetermined height;    -   (d) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (e) after (d), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and a relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers and is thinner than the sample thickness when the        plates are in the open configuration, and the sample is in        contact with the binding site and the storage site;        -   wherein the spacers and plates are configured to make the            thickness of the relevant volume of the sample more uniform            over the area of the relevant volume at the plate closed            configuration than that at the plate open configuration; and            wherein the relevant volume is a portion or an entire volume            of the sample.

UAB6. A device for uniformly a capture-analyte-detection sandwich on abinding site of a plate, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;        -   wherein the first plates has a binding site that has capture            agents being immobilized on the site, and the capture agents            are capable of binding to a target analyte in a sample;        -   wherein the second plate has a storage site that stores the            detection agents, which, are capable of (a) when the storage            site is in contact with the sample, being dissolved into the            sample and diffuse in the sample; and (b) binding to the            target analyte and form a capture agent-target            analyte-detection agent sandwich;        -   wherein one or both of the plates comprise spacers and each            of the spacers is fixed with its respective plate and has a            predetermined height;        -   wherein one of the configurations is an open configuration,            in which: the two plates are either partially or completely            separated apart, the spacing between the plates is not            regulated by the spacers, and the sample is deposited on one            or both of the plates;        -   wherein another of the configuration is a closed            configuration, which is configured after the sample            deposition in the open configuration; and in the closed            configuration: the plates are facing each other, the spacers            and a relevant volume of the sample are between the plates,            the thickness of the relevant volume of the sample is            regulated by the plates and the spacers and is thinner than            the sample thickness when the plates are in the open            configuration, and the sample is in contact with the binding            site and the storage site;        -   wherein the spacers and plates are configured to make the            thickness of the relevant volume of the sample more uniform            over the area of the relevant volume at the plate closed            configuration than that at the plate open configuration; and            wherein the relevant volume is a portion or an entire volume            of the sample.

7.4 Uniform Regulating a Thickness of a Relevant Volume of a SampleBetween Two Plates.

UAB7. A method for regulating a thickness of a relevant volume of asample, comprising:

-   -   (a) obtaining a sample, wherein a thickness of a relevant volume        of the sample is to be regulated;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one or both of the plates        comprise spacers, the spacers have a predetermined inter-spacer        distance and height, and each of the spacers is fixed with its        respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers and is thinner than the maximum thickness of the        sample when the plates are in the open configuration;    -   wherein the spacers and plates are configured to make the        thickness of the relevant volume of the sample more uniform over        the area of the relevant volume at the plate closed        configuration than that at the plate open configuration; and        wherein the relevant volume is a portion or an entire volume of        the sample.

UAB8. A device for regulating a thickness of a relevant volume of asample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations;    -   wherein one or both of the plates comprise spacers, the spacers        have a predetermined inter-spacer distance and height, and each        of the spacers is fixed with its respective plate;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates;    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than the maximum thickness of the sample when the        plates are in the open configuration;    -   wherein the spacers and plates are configured to make the        thickness of the relevant volume of the sample more uniform over        the area of the relevant volume at the plate closed        configuration than that at the plate open configuration; and        wherein the relevant volume is a portion or an entire volume of        the sample

In the methods and the devices in the paragraphs of U1 to U8, theconfiguration of the spacers and plates that make the thickness of therelevant volume of the sample uniform has an embodiment described in thedisclosure.

Uniformity of Sample Thickness.

In the methods and the devices in the paragraphs of U1 to U8, theuniformity of the thickness of the relevant volume of the sample is suchthat the sample thickness at the closed configuration has a relativevariation of at most 0.001%, at most 0.01%, at most 0.05%, at most 0.1%,at most 0.5%, at most 1%, at most 2%, at most 5%, at most 10%, at most20%, at most 30%, at most 50%, at most 75%, at most 90%, less than 100%,or a range between any two of these values.

In a preferred embodiment of the methods and the devices in theparagraphs of U1 to U8, the uniformity of the thickness of the relevantvolume of the sample is such that the sample thickness at the closedconfiguration has a relative variation of at most 0.1%, at most 0.5%, atmost 1%, at most 2%, at most 5%, at most 10%, at most 20%, at most 30%,at most 50%, or a range between any two of these values.

Another parameter that can be important to reduce the saturationincubation time is the uniformity of the sample thickness. If thethickness has a large variation over the binding site, the saturationincubation time can vary from location to location in the binding site,forcing a longer saturation incubation time to ensure all locations inthe binding site having reached the saturation.

8 Amplification Surface

One of current major obstacles for PoC diagnostics and for any assayswhich use a small sample volume is poor sensitivities. It is desirableto enhance the signal of an assay. One aspect of the present inventionis related to the devices and methods that put the binding site on asignal amplification surface (SAS) to amplify the signal for achievinghigher sensitivity. Signal amplification surfaces may also be referredto as signal amplification layers (SAL).

The general structures of SAL comprise nanoscalemetal-dielectric/semiconductor-metal structures, which amplifies localsurface electric field and gradient and light signals. The amplificationare the high at the location where there are the sharp (i.e. largecurvature) edges of a metal structure and the between a small gaps ofthe two metal structures. The highest enhancement regions are thosehaving both the sharp edges and the small gaps. Furthermore, thedimensions for all metallic and non-metallic micro/nanostructuresgenerally are less than the wavelength of the light the SAL amplifies(i.e., subwavelength).

In some embodiments, a SAL layer has as many of the metallic sharp edgesand the small gaps as possible. This requires having a dense group ofmetallic nanostructures with small gaps between the nanostructures. SALstructures may include several different layers. Furthermore, the SALlayer itself can be further improved by a process that can further coverthe portions of the metallic materials that do not have sharp edges andsmall gaps, as described in U.S. provisional application Ser. No.61/801,424, filed on Mar. 15, 2013, and PCT application WO2014197096,filed on Mar. 15, 2014, which are incorporated by reference for allpurposes, as well as PCT/US2014/028417 (Chou et al, “Analyte DetectionEnhancement By Targeted Immobilization, Surface Amplification, AndPixelated Reading And Analysis”), which is incorporated by referenceherein for all purposes.

M1. In some embodiments, the amplification surface is a metal layer onor near the binding site surface. A method for amplifying the signal ofassaying a target entity in a relevant volume of a sample, comprising:

-   -   (a) obtaining a sample that contains a target entity;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one of the plates        comprises, on its surface, one binding site that comprises a        signal amplification surface that is configured to bind the        target entity and to amplify an optical signal which is on or        near the signal amplification surface; and wherein one or both        of the plates comprise spacers and each of the spacers is on its        respective plate and has a predetermined height;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are separated apart and the spacing between the plates is        not regulated by the spacers;    -   (d) after (c), spreading the sample by bringing the plates into        a closed configuration, wherein, in the closed configuration:        the plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers and is thinner than that when the plates are in the        open configuration, and the relevant volume of the sample is in        contact with the binding site; and    -   (e) after (e), incubating, while the plates are in the closed        configuration, for a time period to allow the target entity in        the relevant volume of the sample to bind to the binding site;        wherein the relevant volume is a portion of the sample that        contact to the binding site when the plates are in the closed        configuration.

M2. A device for amplifying the signal in assaying a target entity in arelevant volume of a sample, comprising:

-   -   a first plate and a second plate that are movable relative to        each other into different configurations,    -   wherein the first plate comprises, on its surface, one binding        site, and the binding site comprises a signal amplification        surface that is configured to (i) bind a target entity in a        sample and (ii) amplify an optical signal which is on or near        the signal amplification surface;    -   wherein one or both of the plates comprise spacers and each of        the spacers is on its respective plate and has a predetermined        height;    -   wherein one of the configurations is an open configuration, in        which: the two plates are either partially or completely        separated apart, the spacing between the plates is not regulated        by the spacers, and the sample is deposited on one or both of        the plates,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than that when the plates are in the open        configuration;    -   wherein the relevant volume is a portion of the sample that        contact to the binding site when the plates are in the closed        configuration.

In some embodiments, the signal amplification surface includes at leastone of a metal-dielectric nanostructure, a metal-semiconductornanostructure, and a disk-coupled dots-on-pillar antenna array.

In some embodiments, the signal amplification surface includes a metallayer.

9 Detection and/or Quantification of Volume and/or Concentration (Q)

Quantification and/or control of a relevant volume of a sample is usefulfor quantification and/or control of the concentration of chemicalcompounds (including analytes, entity, reagents, etc.) in the sample.

Common methods for a sample volume quantification include a use of ametered pipette (e.g., Eppendorf's “Research plus pipette, adjustable,0.5-10 μL”, SKU #3120000020), or a geometry. For PoC (point of care) orhome uses, such metering devices are inconvenient to use and/orexpensive. There are needs for simpler and cheaper methods and devicesfor the quantification and/or control of the sample volume and/or theconcentration.

One aspect of the present invention is related to the methods, devices,and systems that quantify and/or control a relevant volume of a samplethat deposited on a plate, without using a metered pipette and/or afixed microfluidic channel. The relevant volume, which can be a portionor the entire volume of the sample, is relevant to the quantificationand/or control of the concentration of target analyte and/or entity inthe sample. The methods, devices and systems in the present inventionare easy to use and low cost.

9.1 A Method for Quantifying a Relevant Volume of a Sample

Q1. A method for quantifying a relevant volume of a sample, comprising:

-   -   (a) obtaining a sample, wherein a relevant volume of the sample        is to be quantified;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one or both of the plates        comprise spacers and the spacers have a predetermined        inter-spacer distance and height, and each of the spacers is        fixed with its respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spread the sample by bringing the plates into a        closed configuration, wherein, in the closed configuration: the        plates are facing each other, the spacers and the relevant        volume of the sample are between the plates, the thickness of        the relevant volume of the sample is regulated by the plates and        the spacers and is thinner than the maximum thickness of the        sample when the plates are in the open configuration, and at        least one of the spacers is inside the sample;    -   (e) quantifying the relevant volume of the sample while the        plates are in the closed configuration;        -   wherein the relevant volume is at least a portion of an            entire volume of the sample.

Q2. In some embodiments, a method for quantifying a relevant volume in asample, comprises:

-   -   (a) obtaining a first plate and a second plate;    -   (b) making a sample to quantified between the two plates;    -   (c) deforming the shape of the sample by compressing the two        plate that reduces the sample thickness and spreading the sample        between the plates laterally; and    -   (d) quantifying the relevant volume of the sample while the        plates are in the closed configuration;    -   wherein the relevant volume is at least a portion of an entire        volume of the sample.

9.2 A Plate for Use in Quantifying a Relevant Volume in a Sample

-   -   Q3. A plate for use in quantifying a relevant volume in a        sample, comprising: a plate that comprises, on its surface, (i)        spacers that have a predetermined inter-spacer distance and        height and are fixed on the surface, and (ii) a sample contact        area for contacting a sample with a relevant volume to be        quantified, wherein at least one of the spacers is inside the        sample contact area.

9.3 A Device for Use in Quantifying a Relevant Volume in a Sample

Q4. A device for quantifying a relevant volume in a sample, comprising:

-   -   a first plate and a second plate that (a) are movable relative        to each other into different configurations and (b) each has a        sample contact area for contacting a sample with a relevant        volume to be quantified,    -   wherein one or both of the plates comprise, on its surface(s),        spacers that have a predetermined inter-spacer distance and        height, and the spacers are fixed with respective plates;    -   wherein one of the configurations is an open configuration, in        which: the two plates are separated apart, the spacing between        the plates is not regulated by the spacers, and the sample is        deposited on one or both of the plates,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than that when the plates are in the open        configuration, and at least one of the spacers is inside the        sample; and    -   wherein the relevant volume of the sample is quantified in the        closed configuration, and the relevant volume is at least a        portion of an entire volume of the sample.

9-5. Measuring a Relevant Volume of a Sample

MS1. In the present invention, the quantifying of a relevant volume ofthe sample while the plates are at a closed configuration includes, butnot limited to, each of the following five embodiments:

-   -   (a) measuring the relevant volume of the sample by a method of        mechanical, optical, electrical, or any combination of thereof;    -   (b) measuring one or several parameter(s) related to the        relevant volume of the sample independently using a method        selected from a method that is mechanical, optical, electrical,        or any combination of thereof;    -   (c) using predetermined one or several parameter(s) related to        the relevant volume of the sample (i.e. the parameter(s) of the        sample determined prior to the plates are at the closed        configuration);    -   (d) determining the relevant volume of the sample by (i)        measuring one or several parameters related to the revel vent        volume when the plates are at a closed configuration and (ii)        predetermining other parameters related to the relevant volume        before the plates are at the closed configuration;    -   (e) determining none-sample volume    -   (f) any combinations of the above (i.e. a, b and c).

In the method of paragraph MS1, the mechanical methods include, but notlimited to, a use of the spacers (i.e. the mechanical device thatregulate the spacing between the inner surfaces of the substrate and thecover-plate to a predetermined value), mechanical probe or rulers, soundwaves (e.g. reflection and/or interference of ultrasound wave to measurethe spacing), or any combination of thereof.

In the method of paragraph MS1, the optical methods include, but notlimited to, a use of light interference, or optical imaging (e.g. takinga 2D (two-dimensional)/3D (three-dimensional) image of the sample,optical imaging of multiple times (with different viewing angles,different wavelength, different phase, and/or different polarization),image processing, or any combination of thereof.

The electrical methods include, but not limited to, capacitive, orresistive or impedance measurements, or any combination of thereof.

In the method of paragraph MS1, in some embodiments, the measurement ofthe sample thickness is to measure the spacing between the innersurfaces of the two plate.

In the method of paragraph MS1, in some embodiments, the use ofpredetermined one or several parameter(s) related to the relevant volumeof the sample, wherein the predetermined parameter is the predeterminedsample thickness that is regulated by the spacers when the plates are ina closed configuration.

In the method of paragraph MS1, in some embodiments, the use ofpredetermined one or several parameter(s) related to the relevant volumeof the sample, wherein the predetermined parameter is the predeterminedthe spacer height.

In the method of paragraph of MS1, in some embodiments, the parametersrelated to the relevant volume of the sample are the parameters at aclosed configuration, that include, but not limited to, (i) the spacingbetween the inner surfaces of the first plate and the second plate (inCROF), (ii) the sample thickness, (iii) the entire or a relevant portionof the sample area, (iv) the entire or a relevant portion of the samplevolume, or (v) any combination of thereof.

In the method of paragraph MS1, in some embodiments, the quantificationof the sample volume or a relevant sample volume, comprising steps of(i) multiplying the sample thickness by the entire sample area to getthe entire sample volume, (ii) multiplying the sample thickness by therelevant sample area to get the relevant sample volume, or (iii)multiplying the relevant sample thickness by the entire or relevantsample area to get the relevant sample volume.

In the method of paragraph MS1, in some embodiments, the measurement isto take 3D (three-dimensional) image of the relevant volume.

In the method of paragraph MS1, in some embodiments, the quantificationof the relevant volume of the sample by measuring the lateral area ofthe relevant volume of the sample, then using it with the thickness ofthe relevant volume to determine the volume of the relevant volume ofthe sample, wherein the thickness of the relevant volume is determinedfrom the information of the spacer, and the information of the spacerinclude the spacer height;

In the method of paragraph MS1, in some embodiments, the quantificationof the relevant volume of the sample by measuring the lateral area ofthe relevant volume of the sample and the spacer together, then using itwith the thickness of the relevant volume and the volume of the spacersto determine the volume of the relevant volume of the sample, whereinthe thickness of the relevant volume is determined from the inform ofthe spacer;

In the method of paragraph MS1, in some embodiments, the quantificationof the relevant volume of the sample by measuring the lateral area andthe thickness of the relevant volume of the sample;

In the method of paragraph MS1, in some embodiments, the quantificationof the relevant volume of the sample by measuring the volume of therelevant volume of the sample optically.

In the method of paragraph MS1, in some embodiments, scale marks areused to assist the quantification of a relevant volume of the samplewhile the plates are at a closed configuration, wherein some embodimentsof the scale markers, their use and measurements, etc. are described inSection 2.

In the method of paragraph MS1, in some embodiments, the quantificationof the relevant volume of the sample comprises a step of substractingthe none-sample volume, wherein the none-sample volume is determined, insome embodiments, by the embodiments described in in the disclosures

9-4. A Method for Quantifying Analytes Concentration in a RelevantVolume of a Sample

Q5. A method for quantifying analytes in a relevant volume of a sample,comprising:

-   -   (a) perform the steps in the method of paragraph Q1; and    -   (b) measuring, after step (a), a signal related to the analytes        from the relevant volume,        -   wherein the relevant volume is at least a portion of an            entire volume of the sample.

Q6. A method for quantifying analytes in a relevant volume of a sample,comprising:

-   -   (a) perform the steps in the method of paragraph Q2; and    -   (b) measuring, after step (a), a signal related to the analytes        from the relevant volume,        -   wherein the relevant volume is at least a portion of an            entire volume of the sample.

In the method of any of paragraphs Q5-6, in some embodiments, it furthercomprises a step of calculating the analytes concentration by dividingthe signal related to the analytes from the relevant volume of thesample by the volume of the relevant volume.

In the method of any of paragraphs Q5-6, one or both plates furthercomprise a binding site, a storage site, or both.

In the method of any of paragraphs Q5-6, in some embodiments, the signalrelated to the analyte is a signal directly from the analytes or a labelattached to the analyte.

Q7. A method for quantifying analytes in a relevant volume of a sample,comprising:

-   -   (a) perform the steps in the method of paragraph Q1, wherein one        or both plates further comprise a binding site; and    -   (b) measuring, after step (a), a signal related to the analytes        from the relevant volume, wherein the relevant volume is at        least a portion of an entire volume of the sample.

Q8. A method for quantifying analytes in a relevant volume of a sample,comprising:

-   -   (a) perform the steps in the method of paragraph Q2, wherein one        or both plates further comprise a binding site; and    -   (b) measuring, after step (a), a signal related to the analytes        from the relevant volume, wherein the relevant volume is at        least a portion of an entire volume of the sample.

In the method of any of paragraphs Q7-8, in some embodiments, the signalrelated to the analyte is a signal directly from the analytes that bindsto the binding site or a label attached to the analyte that binds to thebinding site.

9.5 A Plate for Use in Quantifying Analyte Concentration in a RelevantVolume in a Sample

Q9. A plate for use in quantifying analyte concentration in a relevantvolume in a sample, comprising:

-   -   a plate that comprises, on its surface, (i) spacers that have a        predetermined inter-spacer distance and height, and (ii) a        sample contact area for contacting a sample with analyte        concentration in a relevant volume to be quantified, wherein at        least one of the spacers is inside the sample contact area.

9.6 A Device for Use in Quantifying Analyte Concentration in a RelevantVolume in a Sample

The concentration of target analytes and/or entity in a sample can bequantified or controlled, if the number of target analytes and/or entityin the sample are quantified, as well as the relevant volume of thesample is quantified.

Q10. A device for quantifying analyte concentration in a relevant volumein a sample, comprising:

-   -   a first plate and a second plate that (a) are movable relative        to each other into different configurations and (b) each has a        sample contact area for contacting a sample with analyte        concentration in a relevant volume to be quantified, wherein one        or both of the plates comprise, on its surface(s), spacers that        have a predetermined inter-spacer distance and height, and each        of the spacers are fixed with respective plates;    -   wherein one of the configurations is an open configuration, in        which: the two plates are separated apart, the spacing between        the plates is not regulated by the spacers, and the sample is        deposited on one or both of the plates,    -   wherein another of the configuration is a closed configuration,        which is configured after the sample deposition in the open        configuration; and in the closed configuration: the plates are        facing each other, the spacers and the relevant volume of the        sample are between the plates, the thickness of the relevant        volume of the sample is regulated by the plates and the spacers        and is thinner than that when the plates are in the open        configuration, and at least one of the spacers is inside the        sample; and    -   wherein analyte concentration in the relevant volume of the        sample is quantified in the closed configuration, and the        relevant volume is at least a portion of an entire volume of the        sample.

In the device of any of paragraphs Q9 and Q10, the plate furthercomprises a binding site, or a storage site, or both. One embodiment ofthe binding site is a binding site that bind the analytes in the sample.

In the device of any of paragraphs Q9 and Q10, the plate furthercomprises a or a plurality of scale-markers, wherein some embodiments ofthe scale-markers described in Section 2.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the measuring device includes at least one of an imager anda camera.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the measuring device is configured to image the lateralarea of the relevant volume of the sample.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the measuring device includes a light source to illuminatethe lateral area of the relevant volume of the sample.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the step of calculating the concentration is to divide thetotal target analytes or the entity by the relevant sample volume.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, measuring signal is to use an optical imager to count thenumber of target analytes or entity. For example, the measurement can bea use of optical microscope to measure blood cells (red cell, whitecells, platelets) in a blood sample.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, measuring the number of target analytes or entity in asample can be an embodiment of surface-immobilization assay that catchthe target analytes or the entity on the surface.

In some embodiments, an apparatus for quantifying a volume of a sampleor detecting/quantifying an analyte in a sample comprises any of thedevices in paragraphs Q1-10, plus (1) optical imagers, and/or (2) alight source and optical imagers, etc. The optical imager includes aphotosensor, optical lenses, filters, polarizers, waveplates, beamsplitters, mechanical mounts, or any combination of thereof.

In some embodiments, the measuring of the relevant sample area or volumecomprises (i) having a marker on the first plate, the cover plate,between them, or any combination of thereof, (ii) taking optical imaging(e.g. taking a 2D (two-dimensional)/3D (three-dimensional) image of thesample and the image taking can be multiple times with different viewingangles, different wavelength, different phase, and/or differentpolarization) and (iii) image processing based on the maker and thesample images. The relevant means to be related to the determination oftarget analyte concentration.

Scanning.

In some embodiments, the reading of a signal from a sample uses ascanning method, where a reader (e.g. photodetectors or camera) reads aportion of the sample (or plate) and then moves to another portion ofthe sample (or plate), and such process continues until certainpre-specified port of the sample (or plate) being read. The scan readingof a sample covers all part of the sample (or the plate) or a fractionof the sample (or the plate). In some embodiments, the scan reading areassisted by the location markers that indicate a location of the sample(or the plate). One example of the location markers is the periodicspacers, which has a fixed period and location, or the markers for therelevant area which also has predetermined location and size forindicating a location of the sample or plate.

10 Detection and Quantification of Analytes and Others (D)

In certain embodiments, an analyte is detected and/or quantified (i.e.assayed) by measuring a signal related to the analyte, wherein thesignal is an optical signal, electrical signal, mechanical signal,chemi-physical signal, or any combination of thereof. In someembodiments, the analyte assaying are performed when the two plates in aCROF device are close to each other. In some embodiments, the analyteassaying are performed when the two plates in a CROF device areseparated from each other.

The optical signal includes, but not limited to, light reflection,scattering, transmission, absorption, spectrum, color, emission,intensity, wavelength, location, polarization, luminescence,fluorescence, electroluminescence, chemoluminescence,eletrochemoluminescence, or any combination of thereof. The opticalsignal is in the form of optical image (i.e. light signal vs location ofthe sample or device) or a lump sum of all photons coming from a givenarea or volume. A preferred wavelength of the light is in a range of 400nm to 1100 nm, a range of 50 nm to 400 nm, a range of 1 nm to 50 nm, ora range of 1100 to 30,000 nm. Another preferred wavelength is interahertz.

The electrical signal includes, but not limited to, charge, current,impedance, capacitance, resistance, or any combination of thereof. Themechanical signal includes, but not limited to, mechanical wave, soundwave, shock wave, or vibration. The chemi-physical signal includes, butnot limited to, PH value, ions, heat, gas bubbles, color change, thatare generated in an reaction.

For example, the label is a bead and the label is attached to the labelthrough an analyte specific binding process (e.g. use detection agent tobind the bead to the analyte, use capture agent to capture the analytewith bead, use a capture agent to bind the analyte and then usedetection agent to attach the bead, or other approaches. Note thecapture and detection agents bind the analyte specifically), then ameasurement is used to identify each of the beads that are attached tothe analytes, and count them.

In some embodiments, each of the analyte or the beads are sensed andcounted by optical means (such as (i) optical labels and reading of thelabels, (ii) surface plasmon resonance, (iii) optical interferences,(iv) electrical methods (e.g. capacitance, resistance, impedance, etc.),or others. The sensors can be on the surface of the first plate and/orthe second plate.

Certain embodiments may include determining the analyte concentration in(a) surface immobilization assay, (b) bulk assay (e.g., blood cellcounting), and (c) others. In some embodiments, the methods of thesample volume, the relevant volume of the sample, or the concentrationuses a smart-phone.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the measuring a signal is to measure the number of theanalytes in the sample, or measure the number of a label being attachedto the analytes in the sample. In another embodiment of paragraph Q5,the “measuring signal” is to (a) identify each of the analyte or thelabel attached to each analyte, and (b) count their number.

In some embodiments, the analytes detection is an electrical method whenelectrodes are put on one or both of the first and second plates (thisapplies to any of the methods and devices that uses CROF). Theelectrodes measure the charge, current, capacitance, impedance, orresistance of a sample, or any combination of thereof. The electrodesmeasure an electrolyte in a sample. The electrodes have a thicknessequal or less than the thickness spacer. In some embodiments, theelectrode serve as a part of the spacers. The electrodes are made ofvarious conducting materials. A preferred electrode material is gold,silver, aluminum, copper, platinum, carbon nanotubes, or any combinationof thereof.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the measuring uses the devices that is a camera orphotodetector plus an optional processor configured to make themeasurement.

In the method or the device of any of paragraphs of Q1-10, in someembodiments, the concentration determining devices comprises a processorconfigured to determine the concentration from the measurements (volume,area, thickness, number of analytes, intensity)

In the method or the device of any of paragraphs of Q1-10, in someembodiments, it further comprising a concentration determining device isconfigured to determine the concentration of the target analytes in therelevant volume from the measured lateral area, the thickness, and themeasured amount of the target molecules.

More on Signal Detection Using Pixelated Reading and Analysis

In present invention, in some embodiments, the signals from the sample,analytes, and entity, binding sites, reagents, CROF plates, or anycombinations of thereof are detected and analytes. Some embodiments ofthe signal detection using pixelated reading and analysis are describedin the disclosure, while some other embodiments are described inPublication Number: WO2014144133 A and Application Number:PCT/US2014/028417 (Chou et al, “Analyte Detection Enhancement ByTargeted Immobilization, Surface Amplification, And Pixelated ReadingAnd Analysis”), which is incorporated by reference herein for allpurposes.

In some embodiments, the signal is electromagnetic signal, includingelectrical and optical signals with different frequencies, lightintensity, fluorescence, chromaticity, luminescence (electrical andchemo-luminescence), Raman scattering, time resolved signal (includingblinking). The signals also can be the forces due to local electrical,local mechanical, local biological, or local optical interaction betweenthe plate and the reading device. The signal also includes the spatial(i.e. position), temporal and spectral distribution of the signal. Thedetection signal also can be absorption.

The analyte include proteins, peptides, DNA, RNA, nucleic acid, smallmolecules, cells, nanoparticles with different shapes. The targetedanalyte can be either in a solution or in air or gas phase. The sensingincludes the detection of the existence, quantification of theconcentration, and determination of the states of the targeted analyte.

In some embodiments, electric field is used to assist molecularselectivity, or bonding, and detection.

Detection/Reading Methods

In some embodiments of optical detection (i.e. detection byelectromagnetic radiation), the methods include, but not limited to,far-field optical methods, near-field optical methods, epi-fluorescencespectroscopy, confocal microscopy, two-photon microscopy, and totalinternal reflection microscopy, where the target analytes are labelledwith an electromagnetic radiation emitter, and the signal in thesemicroscopies can be amplified by the amplification surface of a CROFplate.

In some embodiments, the signal comprises the information of theposition, local intensity, local spectrum, local polarization, localphase, local Raman signature of said signals, or any combination ofthereof.

In some embodiments, the detection of a signal is to measure a lump-sumsignal from an area (i.e. the signal from the area, regardless whichlocation in the area).

In certain embodiments, the detection of signal is to measure an signalimage of an area (i.e. signal vs location); namely, the area is dividedinto pixels and the signal from each pixel of the area is individuallymeasured, which is also termed “PIX” or “pixelated imaging detection”.The individual measurement of each pixel can be in parallel orsequential or a mix.

In some embodiments, the reading uses appropriate detecting systems forthe signal to be detected in sequence or in parallel or theircombination. In a sequential detection, one or several pixels aredetected a time, and scanner will be used to move the detection intoother areas of the SAL. In a parallel detection, a multipixel detectorarray, such as imaging camera (e.g. CCD's), will be used to take detectthe signals from different pixels at the same time. The scan can besingle path or multi-path with a different pixel size for each path.FIG. 2C of PCT/US2014/028417 schematically illustrates pixelated readingon an x, y, z stage.

The pixel size for the reading/detection will be adjusted to for thebalance of optical resolution and total reading time. A smaller pixelsize will take a longer time for reading/scanning the entire or fractionof the SAL. A typical pixel size is 1 um to 10 um in size. The pixel hasdifferent shapes: round, square and rectangle. The lower limit of thepixel size is determined by the optical resolution of the microscopesystem, and the higher limit of the pixel size is determined in order toavoid reading error from the uneven optical response of the imager(optical aberration, illumination uniformity, etc.).

Reading System

Referred to the Figures in of PCT/US2014/028417, an embodiment of areading system comprises (a) a plate or plates used for CROF; (b) areading device 205 for producing an image of signals emanating from asurface of said plate, wherein signals represent individual targetedanalyte binding events; (c) a device assembly 300 that holds the plateand the imager; (d) an electronics and a data storage 301 for storingsaid image; and (e) a computer comprising programming for identifyingand counting individual binding events in an area of the image.

The device assembly 300 controls or changes the relative positionbetween the plate and the reading device, in at least one of the three(x, y, z) orthogonal directions, for reading the signal. An embodimentof the device assembly comprises a scanner 301. In some embodiments, thescanner 301 scans in at least one of the three (x, y, z) orthogonaldirections.

In some embodiments, the reading device 302 is a CCD camera. In someembodiments, the reading device 302 is a photodetector comprising one ormore other optical devices that are selected from optical filters 303,spectrometer, lenses 304, apertures, beam splitter 305, mirrors 306,polarizers 307, waveplates, and shutters. In some embodiments, thereading device 302 is a smartphone or mobile phone, which have thecapability of local and remote communications. The reading devicecollects the position, local intensity, local spectrum, local Ramansignature of said signals, or any combination of thereof.

In some embodiments, optical filters 303, light beam splitters 305,optical fibers, a photodetector (e.g. pn junction, a diode, PMT(photomultiplier tube), or APD (Avalanch Photo Diode), imaging camera(e.g. CCD's, or cellphone camera) and spectrometer together with ascanner provided by the device assembly 301 are coupled to a microscopesystem which uses a far-field confocal setting or a wide-field viewsetting.

In some embodiments, in confocal setting, the reading is performed byrecording the brightness, temporal change and spectral change of one ora few pixels a time and raster scanning the entire interested area ofthe SAL. In some embodiments, in wide-field view setting, a camera isused to record the brightness and temporal change of the entire or afraction of SAL area a time. In some embodiments, proper optical filtersand light beam manipulators (polarizer, beam splitters, optical fibers,etc.) is need to ensure only the desired signal is collected anddetected. FIG. 9 of PCT/US2014/028417 schematically illustrates onearrangement of components for this system. In some embodiments, theanalysis comprises of an imaging processing methods, including, notlimited to, the methods in Open-CV or Image-J.

Pixelated Analysis (PIX).

In some embodiments of PIX, the signals detected in a pixelated mannerare analyzed to determine the number and/or types of the particularmolecules at a particular pixel or several pixels, which, in turn isused to quantify the type and/or concentration of the targeted analytes.The term “signal detected in a pixelated manner” refers to the methodwhere the area that has signal(s) is divided into pixels and the signalfrom each pixel of the area is individually measured, which is alsotermed “PIX” or “pixelated imaging detection”. The individualmeasurement of each pixel can be in parallel or sequential or a mix.

In some embodiments, the analysis comprises to analyze the spatial,tempo, spectral information of the signal. In some embodiments, theanalysis include, but not limited to, statistical analysis, comparison,integration, and others. FIG. 5 of PCT/US2014/028417 shows a flow chartfor one embodiment of this method.

11 Labels

One or any combinations of the embodiments of the optical labelsdescribed in the entire disclosure applies to all the methods anddevices described in the entire description of the present invention.

In some embodiments, a label(s) is attached to a detection agent(s), ananalyte(s) or an entity (ties). In certain embodiments, the label is anoptical label, an electric label, enzymes that can be used to generatean optical or electrical signal, or any combination of thereof. Incertain embodiments, a detection agent(s), an analyte(s) or an entity(ties) are attached a connection molecule (e.g. protein, nucleic acid,or other compounds) which later is attached to a label. In certainembodiments, cells (e.g. blood cells, bacteria, etc.) or nanoparticlesare stained by a labels. In some embodiments, an optical label is anobject that can generate an optical signal, wherein the generation ofthe optical signal includes, but not limited to, light (i.e. photon's)reflection, scattering, transmission, absorption, spectrum, color,emission, intensity, wavelength, location, polarization, luminescence,fluorescence, electroluminescence, photoluminescence (fluorescence),chemoluminescence, electrochemiluminescence, or any combination ofthereof. In some embodiments, the optical signal is in the form ofoptical image (i.e. light signal vs location of the sample or device) ora lump sum of all photons coming from a given area or volume. Apreferred wavelength of the light is in a range of 400 nm to 1100 nm, arange of 50 nm to 400 nm, a range of 1 nm to 50 nm, or a range of 1100to 30,000 nm. Another preferred wavelength is in terahertz.

Beads, Nanoparticles, and Quantum Dots.

In some embodiments, the optical label is beads, nanoparticles, quantumdots, or any combination of thereof.

In some embodiments, the diameter of the bead, nanoparticles, or quantumdots is 1 nm or less, 2 nm or less, 5 nm or less, 10 nm or less, 20 nmor less, 30 nm or less, 40 nm or less, 50 nm or less, 60 nm or less, 70nm or less, 80 nm or less, 100 nm or less, 120 nm or less, 200 nm orless, 300 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less,1500 nm or less, 2000 nm or less, 3000 nm or less, 5000 nm or less, or arange between any two of the values.

In some embodiments, the beads or quantum dots are used as labels andthey are precoated on the plates of CROF and the inner spacing betweenthe two plates are 1 um or less, 10 um or less, 50 um or less, or arange between any two of the values.

In some embodiment, the separation between the beads in a solution

-   -   Diffusion time. (The thickness of the relevant volume of the        transfer medium leads to the diffusion time of an optical label        across the thickness, to be less than 1 ms,    -   The dissolving time can controlled. The control can use photon,        heat or other exications and their combinations. The dissolving        will not start until an excitation energy is applied. In some        embodiments of the label are nanoparticles that has a diameter        of 10 nm or larger.        The nanoparticles of such large diameter has less diffusion        constant than small molecules (mass <1000 Da) and large        molecules (mass=1,000 to 1,000,000 Dalton (da), leading to a        longer diffusion time for a given solution and distance. To        reduce the diffusion time, is to reduce the diffusion distance.

They have particular advantages over the prior art, when the opticallabels are beads or other nanoparticles that have a diameter large thana few nanometers. This is because that the diffusion constant of anobject in a liquid is, for the first order approximation, inverselyproportional to the diameter of the object (according to Einstein-Stokesequation).

For example, a bead optical label with a diameter of 20 nm, 200, and2000 nm respectively has a diffusion constant and hence a diffusion time10, 100, and 1000 times larger and longer than that for a bead of 2 nm.For a typical diffusion distance used in current assays, this would leadto a long saturation incubation time that is in practical for PoC (Pointof Care) applications.

However, the present invention has solved the long incubation time foroptical labels with a diameter larger than a few nanometers. The presentinvention has the optical label stored on a plate surface, and thenplaces the storage surface next to binding site with a separate distance(between the two) in sub-millimeter, microns or even nanometer scale andfill the separation gap by a transfer medium (where the stored opticallabel dissolved into the transfer medium and diffuse to the bindingsite). The present invention also able to control such small distanceuniformly over large binding site area and easily by using spacertechnologies.

Labeling the analyte may include using, for example, a labeling agent,such as an analyte specific binding member that includes a detectablelabel. Detectable labels include, but are not limited to, fluorescentlabels, colorimetric labels, chemiluminescent labels, enzyme-linkedreagents, multicolor reagents, avidin-streptavidin associated detectionreagents, and the like. In certain embodiments, the detectable label isa fluorescent label. Fluorescent labels are labeling moieties that aredetectable by a fluorescence detector. For example, binding of afluorescent label to an analyte of interest may allow the analyte ofinterest to be detected by a fluorescence detector. Examples offluorescent labels include, but are not limited to, fluorescentmolecules that fluoresce upon contact with a reagent, fluorescentmolecules that fluoresce when irradiated with electromagnetic radiation(e.g., UV, visible light, x-rays, etc.), and the like.

In certain embodiments, suitable fluorescent molecules (fluorophores)for labeling include, but are not limited to, IRDye800CW, Alexa 790,Dylight 800, fluorescein, fluorescein isothiocyanate, succinimidylesters of carboxyfluorescein, succinimidyl esters of fluorescein,5-isomer of fluorescein dichlorotriazine, cagedcarboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine,Texas Red, propidium iodide, JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanineiodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (tetramethylrhodamine methyl ester), TMRE (tetramethyl rhodamine ethyl ester),tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine,green fluorescent protein, blue-shifted green fluorescent protein,cyan-shifted green fluorescent protein, red-shifted green fluorescentprotein, yellow-shifted green fluorescent protein,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives, such as acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propionicacid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives:coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriaamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2-,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-(dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)amino-fluorescein (DTAF),2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelli-feroneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl hodamine isothiocyanate (TRITC); riboflavin;5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CALFluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7;IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine,coumarins and related dyes, xanthene dyes such as rhodols, resorufins,bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazidessuch as luminol, and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, fluorescent europium and terbium complexes;combinations thereof, and the like. Suitable fluorescent proteins andchromogenic proteins include, but are not limited to, a greenfluorescent protein (GFP), including, but not limited to, a GFP derivedfrom Aequoria victoria or a derivative thereof, e.g., a “humanized”derivative such as Enhanced GFP; a GFP from another species such asRenilla reniformis, Renilla mulleri, or Ptilosarcus guemyi; “humanized”recombinant GFP (hrGFP); any of a variety of fluorescent and coloredproteins from Anthozoan species; combinations thereof; and the like.

In certain embodiments, the dyes can be used to stain the blood cellscomprise Wright's stain (Eosin, methylene blue), Giemsa stain (Eosin,methylene blue, and Azure B), May-Grünwald stain, Leishman's stain(“Polychromed” methylene blue (i.e. demethylated into various azures)and eosin), Erythrosine B stain (Erythrosin B), and other fluorescencestain including but not limit to Acridine orange dye,3,3-dihexyloxacarbocyanine (DiOC6), Propidium Iodide (PI), FluoresceinIsothiocyanate (FITC) and Basic Orange 21 (BO21) dye, Ethidium Bromide,Brilliant Sulfaflavine and a Stilbene Disulfonic Acid derivative,Erythrosine B or trypan blue, Hoechst 33342, Trihydrochloride,Trihydrate, and DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride).

In certain embodiments, the labeling agent is configured to bindspecifically to the analyte of interest. In certain embodiments, alabeling agent may be present in the CROF device before the sample isapplied to the CROF device. In other embodiments, the labeling agent maybe applied to the CROF device after the sample is applied to the CROFdevice. In certain embodiments, after the sample is applied to the CROFdevice, the CROF device may be washed to remove any unbound components,e.g. un bound analyte and other non-analyte coponents in the sample, andthe labeling agent may be applied to the CROF device after the washingto label the bound analyte. In some embodiments, the CROF device may bewashed after the labeling agent is bound to the analyte-capture agentcomplex to remove from the CROF device any excess labeling agent that isnot bound to an analyte-capture agent complex.

In certain embodiments, the analyte is labeled after the analyte isbound to the CROF device, e.g., using a labeled binding agent that canbind to the analyte simultaneously as the capture agent to which theanalyte is bound in the CROF device, i.e., in a sandwich-type assay.

In some embodiments, a nucleic acid analyte may be captured on the CROFdevice, and a labeled nucleic acid that can hybridize to the analytesimultaneously as the capture agent to which the nucleic acid analyte isbound in the CROF device.

In certain aspects, a CROF device enhances the light signal, e.g.,fluorescence or luminescence, that is produced by the detectable labelbound directly or indirectly to an analyte, which is in turn bound tothe CROF device. In certain embodiments, the signal is enhanced by aphysical process of signal amplification. In some embodiments, the lightsignal is enhanced by a nanoplasmonic effect (e.g., surface-enhancedRaman scattering). Examples of signal enhancement by nanoplasmoniceffects is described, e.g., in Li et al, Optics Express 2011 19:3925-3936 and WO2012/024006, which are incorporated herein by reference.In certain embodiments, signal enhancement is achieved without the useof biological/chemical amplification of the signal. Biological/chemicalamplification of the signal may include enzymatic amplification of thesignal (e.g., used in enzyme-linked immunosorbent assays (ELISAs)) andpolymerase chain reaction (PCR) amplification of the signal. In otherembodiments, the signal enhancement may be achieved by a physicalprocess and biological/chemical amplification.

Sensitivity.

In certain embodiments, the CROF device is configured to have adetection sensitivity of 0.1 nM or less, such as 10 pM or less, or 1 pMor less, or 100 fM or less, such as 10 fM or less, including 1 fM orless, or 0.5 fM or less, or 100 aM or less, or 50 aM or less, or 20 aMor less. In certain embodiments, the CROF device is configured to have adetection sensitivity in the range of 10 aM to 0.1 nM, such as 20 aM to10 pM, 50 aM to 1 pM, including 100 aM to 100 fM. In some instances, theCROF device is configured to be able to detect analytes at aconcentration of 1 ng/mL or less, such as 100 pg/mL or less, including10 pg/mL or less, 1 pg/mL or less, 100 fg/mL or less, 10 fg/mL or less,or 5 fg/mL or less. In some instances, the CROF device is configured tobe able to detect analytes at a concentration in the range of 1 fg/mL to1 ng/mL, such as 5 fg/mL to 100 pg/mL, including 10 fg/mL to 10 pg/mL.In certain embodiments, the CROF device is configured to have a dynamicrange of 5 orders of magnitude or more, such as 6 orders of magnitude ormore, including 7 orders of magnitude or more.

Reading.

In certain instances, the period of time from applying the sample to theCROF device to reading the CROF device may range from 1 second to 30minutes, such as 10 seconds to 20 minutes, 30 seconds to 10 minutes,including 1 minute to 5 minutes. In some instances, the period of timefrom applying the sample to the signal enhancing detector to generatingan output that can be received by the device may be 1 hour or less, 30minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes orless, 3 minutes or less, 1 minute or less, 50 seconds or less, 40seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds orless, 5 seconds or less, 2 seconds or less, 1 second or less, or evenshorter. In some instances, the period of time from applying the sampleto the signal enhancing detector to generating an output that can bereceived by the device may be 100 milliseconds or more, including 200milliseconds or more, such as 500 milliseconds or more, 1 second ormore, 10 seconds or more, 30 seconds or more, 1 minute or more, 5minutes or more, or longer.

Any suitable method may be used to read the CROF device to obtain ameasurement of the amount of analyte in the sample. In some embodiments,reading the CROF device includes obtaining an electromagnetic signalfrom the detectable label bound to the analyte in the CROF device. Incertain embodiments the electromagnetic signal is a light signal. Thelight signal obtained may include the intensity of light, the wavelengthof light, the location of the source of light, and the like. Inparticular embodiments, the light signal produced by the label has awavelength that is in the range of 300 nm to 900 nm. In certainembodiments, the light signal is read in the form of a visual image ofthe CROF device.

In certain embodiments, reading the CROF device includes providing asource of electromagnetic radiation, e.g., light source, as anexcitation source for the detectable label bound to the biomarker in theCROF device. The light source may be any suitable light source to excitethe detectable label. Exemplary light sources include, but are notlimited to, sun light, ambient light, UV lamps, fluorescent lamps,light-emitting diodes (LEDs), photodiodes, incandescent lamps, halogenlamps, and the like.

Reading the CROF device may be achieved by any suitable method tomeasure the amount of analyte that is present in the sample and bound tothe CROF device. In certain embodiments, the CROF device is read with adevice configured to acquire the light signal from the detectable labelbound to the analyte in the CROF device. In some cases, the device is ahandheld device, such as a mobile phone or a smart phone. Any suitablehandheld device configured to read the CROF device may be used in thedevices, systems and methods in the present invention. Cerain deviceembodiments configured to read the CROF device are described in, e.g.,U.S. Provisional Application Ser. No. 62/066,777, filed on Oct. 21,2014, which is incorporated herein by reference.

In some embodiments, the device includes an optical recording apparatusthat is configured to acquire a light signal from the CROF device, e.g.,acquire an image of the CROF device. In certain instances, the opticalrecording apparatus is a camera, such as a digital camera. The term“digital camera” denotes any camera that includes as its main componentan image-taking apparatus provided with an image-taking lens system forforming an optical image, an image sensor for converting the opticalimage into an electrical signal, and other components, examples of suchcameras including digital still cameras, digital movie cameras, and Webcameras (i.e., cameras that are connected, either publicly or privately,to an apparatus connected to a network to permit exchange of images,including both those connected directly to a network and those connectedto a network by way of an apparatus, such as a personal computer, havingan information processing capability). In one example, reading the CROFdevice may include video imaging that may capture changes over time. Forexample, a video may be acquired to provide evaluation on dynamicchanges in the sample applied to the CROF device.

In certain embodiments, the optical recording apparatus has asensitivity that is lower than the sensitivity of a high-sensitivityoptical recording apparatus used in research/clinical laboratorysettings. In certain cases, the optical recording apparatus used in thesubject method has a sensitivity that is lower by 10 times or more, suchas 100 times or more, including 200 times or more, 500 times or more, or1,000 times or more than the sensitivity of a high-sensitivity opticalrecording apparatus used in research/clinical laboratory settings.

In certain embodiments, the device may have a video display. Videodisplays may include components upon which a display page may bedisplayed in a manner perceptible to a user, such as, for example, acomputer monitor, cathode ray tube, liquid crystal display, lightemitting diode display, touchpad or touchscreen display, and/or othermeans known in the art for emitting a visually perceptible output. Incertain embodiments, the device is equipped with a touch screen fordisplaying information, such as the image acquired from the detectorand/or a report generated from the processed data, and allowinginformation to be entered by the subject.

12 Multiplexing

In any embodiment described herein, the system may be designed forperforming a multiplex assay and, as such, may contain multiple storagesites, multiple binding sites, or multiple storage sites and multiplebinding sites such that different assays can be performed on differentareas on the surface of one of the plates. For example, in oneembodiment, in one embodiment, one of the plates may contain multiplebinding site that each contain a different capture agent, therebyallowing the detection of multiple analytes in the sample in the sameassay. The sites may be spatially separated from, although proximal to,one another.

FIG. 16 schematically illustrates an exemplary embodiment of the presentinvention, a multiplexed detection in a single CROF device using onebinding site one plate and a plurality of storage sites on the otherplate. Panel (a) and (b) is a perspective and a cross-sectional view ofan exemplary device, respectively. In the exemplary case, themultiplexed CROF device comprises a first plate and a second plate,wherein one surface of the first plate has one binding site; wherein onesurface of the second plate has a plurality of storage sites; andwherein different storage sites can have the same detection agent but ofdifferent concentrations or can have different detection agents of thesame or different concentrations. In some embodiments, the area of thebinding site is larger that of each storage site. In some embodiments,the binding site area is larger than the total area of all storagesites, and/or the binding site area is aligned with the storage sites(i.e. they are top each other, namely, the shortest distance between thebinding site and a point on the storages are the same or nearly thesame).

FIG. 17 schematically illustrates a further exemplary embodiment of thepresent invention, a multiplexed detection in a single CROF device usingone storage site on one plate and multiple binding sites on the otherplate. Panel (a) and (b) is a perspective and a cross-sectional view ofan exemplary device, respectively. In the exemplary case, themultiplexed CROF device comprises a first plate and a second plate,wherein one surface of the first plate has multiple binding sites;wherein one surface of the second plate has one storage site; andwherein different binding sites can have the same capture agent but ofdifferent concentrations or can have different capture agents of thesame or different concentrations. In some embodiments, the area of thestorage site is larger that of each storage site. In some embodiments,the storage site area is larger than the total area of all bindingsites, and/or is aligned with the binding sites (i.e. they are top eachother).

FIG. 18 schematically illustrates a further exemplary embodiment of thepresent invention, a multiplexed detection in a single CROF device withmultiple binding sites on one plate and multiple corresponding storagesites on another plate. Panel (a) and (b) is a perspective and across-sectional view of an exemplary device, respectively. In theexemplary case, a multiplexed CROF device comprises a first plate and asecond plate, wherein one surface of the first plate has a plurality ofbinding sites; wherein one surface of the second plate has a pluralityof corresponding storage sites; wherein each corresponding storage siteis located in a location on the second plate that is corresponding tothe location of a binding site on the first plate, so that when theplates are placed face-to-face, each binding site overlaps with only onestorage site and each storage site overlaps with only one storage site;wherein different storage sites can have the same detection agent but ofdifferent concentrations or can have different detection agents of thesame or different concentrations; and wherein different storage sitescan have the same capture agent but of different concentrations or canhave different capture agents of the same or different concentrations.

In certain embodiments, the device of any of FIGS. 10, 11, and 12,wherein the first plate further comprises, on its surface, a firstpredetermined assay site and a second predetermined assay site, whereinthe distance between the edges of the neighboring multiple assay sitesis substantially larger than the thickness of the uniform thicknesslayer when the plates are in the closed position, wherein at least apart of the uniform thickness layer of the sample is over thepredetermined assay sites, and wherein the sample has one or a pluralityof analytes that are capable of diffusing in the sample. By making thedistance between the edges of the neighboring multiple assay sites largethan the sample thickness, it makes it possible to have multiple bindingsites without fluidically isolated the different portion of a sample,since an saturation incubation of the assay can complete between asignificant inter-diffusion between the two neighboring sites. Byproperly choosing the ratio of the neighboring distance to the samplethickness and properly selecting the measurement time between a timelonger than the assay saturation incubation time but less than a timefor a significant inter-diffusion between two neighboring sites, one cando multiplexing by CROF without isolating different part of a sample. Insome embodiments, the ratio of the neighbor distance to the samplethickness at the closed configuration is 1.5 or larger, 3 or larger, 5or larger, 10 or larger, 20 or larger, 30 or larger, 50 or larger, 100or larger, 200 or larger, 1000 or larger, 10,000 or larger, or a rangebetween any two of the values. The ratio is 3 or larger for a preferredembodiment, 5 or larger for another preferred embodiment, 10 or largerfor a certain preferred embodiment, 30 or larger for another preferredembodiment, and 100 or larger for another preferred embodiment.

In certain embodiments, the device of any of FIGS. 10, 11, and 12,wherein the first plate has, on its surface, at least three analyteassay sites, and the distance between the edges of any two neighboringassay sites is substantially larger than the thickness of the uniformthickness layer when the plates are in the closed position, wherein atleast a part of the uniform thickness layer is over the assay sites, andwherein the sample has one or a plurality of analytes that are capableof diffusing in the sample.

In certain embodiments, the device of any of FIGS. 10, 11, and 12,wherein the first plate has, on its surface, at least two neighboringanalyte assay sites that are not separated by a distance that issubstantially larger than the thickness of the uniform thickness layerwhen the plates are in the closed position, wherein at least a part ofthe uniform thickness layer is over the assay sites, and wherein thesample has one or a plurality of analytes that are capable of diffusingin the sample.

The method or the devices of any of paragraph of U1-6, X-6, P1-8, W1-6,V1-4, UAB1-8, M1-2, S1-2, Q110, and H1 as well as their any combination,wherein the first and second plate further comprise the binding site(s)and the storage site, as described in FIG. 10, FIG. 11, or FIG. 12 formultiplexed detection.

In these embodiments the device may for parallel, multiplex, assaying ofa liquid sample without fluidic isolation (i.e., without their being aphysical barrier between the assay regions). This device may comprise afirst plate and a second plate, wherein: i. the plates are movablerelative to each other into different configurations; one or both platesare flexible; ii. one or both of the plates comprise spacers that arefixed with a respective plate; and the spacers have a predeterminedsubstantially uniform height and a predetermined constant inter-spacerdistance; iii. each of the plates has, on its respective surface, asample contact area for contacting a sample that contains a sample thatcontains one or more target analytes which is capable of diffusing inthe sample, iii. the first plate has, on its surface, one or a pluralityof binding sites that each has a predetermined area comprising a captureagent that binds and immobilizes a corresponding target analyte of thesample; and iv the second plate has, on its surface, one or a pluralityof corresponding storage sites that each has a predetermined area andcomprises a detection agent of a concentration that, upon contacting thesample, dissolves into the sample and diffuses in the sample, whereineach capture agent, target analyte and corresponding detection agent iscapable of forming a capture agent-target analyte-detection agentsandwich in a binding site of the first plate; wherein one of theconfigurations is an open configuration, in which: the two plates areeither partially or completely separated apart, the spacing between theplates is not regulated by the spacers, and the sample is deposited onone or both of the plates, and wherein another of the configurations isa closed configuration which is configured after the sample depositionin the open configuration; and in the closed configuration: i. at leastpart of the sample is compressed into a layer of uniform thickness thatis in contact with and confined by the inner surfaces of the two platesand that covers the one or a plurality of binding sites and the one or aplurality of storage sites, ii the one or a plurality of correspondingstorage sites are over the one or a plurality of binding sites, and iii.the uniform thickness of the layer is regulated by the spacers and theplates, is less than 250 um, and is substantially less than the lineardimension of the predetermined area of each storage site; and iv. thereis no fluidic isolation between the binding site and/or the storagesites, wherein the separation between the edges of the neighboringstorage sites and the separation between the edges of the neighboringbinding sites are larger than the distance that a target analyte ordetection agent can diffuse in the relevant time, and wherein there isno fluidic isolation between the binding site sites and/or the storagesites.

In some embodiments, the first plate has, on its surface, a plurality of(at least 2, at least 4 or at least 16 or more) of the binding sites.

In some embodiments, each of said plurality of binding sites binds to adifferent target analyte.

In some embodiments, the second plate has, on its surface, a plurality(at least 2, at least 4 or at least 16 or more) of the correspondingstorage sites.

In some embodiments, each of the plurality of corresponding storagesites binds to a different target analyte.

In some embodiments, the first plate has, on its surface, a plurality ofsaid binding sites and the second plate has, on its surface, a pluralityof said corresponding storage sites, wherein each binding site faces acorresponding storage site when the plates are in the closedconfiguration.

In some embodiments, the first plate has, on its surface, a plurality ofsaid binding sites and the second plate has, on its surface, a storagesite, wherein at least some of the binding sites face an area in thestorage site when the plates are in the closed configuration.

In some embodiments the first plate has, on its surface, a binding siteand the second plate has, on its surface, a plurality of storage sites,wherein at least some of the storage sites face an area in the bindingsite when the plates are in the closed configuration.

In some embodiments the first plate has, on its surface, a plurality ofbinding sites, wherein the binding sites contain different captureagents that bind and immobilize the same target analyte.

In some embodiments the first plate has, on its surface, a plurality ofbinding sites, wherein the binding sites contain the same capture agent.

In some embodiments, the capture agent is at different densities in thedifferent binding sites. These embodiments may be used to provide a wayto quantify the amount of analyte in a sample.

In some embodiments, there is a separation between two neighboringbinding sites or two neighboring storage sites, and the ratio of theseparation to the sample thickness in the closed configuration is atleast 3, e.g., at least 5, at least 10, at least 20 or at least 50.

In some embodiments, the inter-spacer distance is in the range of 1 umto 120 um.

In some embodiments, the flexible plates have a thickness in the rangeof 20 um to 250 um (e.g., in the range of 50 um to 150 um) and Young'smodulus in the range 0.1 to 5 GPa (e.g., in the range of 0.5-2 GPa).

In some embodiments, the thickness of the flexible plate times theYoung's modulus of the flexible plate is in the range 60 to 750 GPa-um.

In some embodiments, this method may comprise (a) obtaining a samplethat contains one or more target analytes, which are capable ofdiffusing in the sample; (b) obtaining a first and second plates thatare movable relative to each other into different configurations,wherein: i. one or both of the plates comprise spacers that are fixedwith a respective plate and one or both plates are flexible, ii. thespacers have a predetermined substantially uniform height and apredetermined constant inter-spacer distance, iii. the first plate has,on its surface, one or a plurality of binding sites that each has apredetermined area comprising a capture agent that binds and immobilizesa corresponding target analyte of (a); and iv. the second plate has, onits surface, one or a plurality of corresponding storage sites that eachhas a predetermined area and comprises a detection agent of aconcentration that, upon contacting the sample, dissolves into thesample and diffuses in the sample, wherein each capture agent, targetanalyte and corresponding detection agent is capable of forming acapture agent-target analyte-detection agent sandwich in a binding siteof the first plate; (c) depositing the sample on one or both of theplates when the plates are configured in an open configuration, whereinthe open configuration is a configuration in which the two plates areeither partially or completely separated apart and the spacing betweenthe plates is not regulated by the spacers; (d) after (c), compressingthe sample by bringing the two plates into a closed configuration,wherein the closed configuration is a configuration in which: i. atleast part of the sample is compressed into a layer of uniform thicknessthat is in contact with and confined by the inner surfaces of the twoplates and that is in contact with the one or a plurality of bindingsites and the one or a plurality of storage sites, ii the one or aplurality of corresponding storage sites are over the one or a pluralityof binding sites, and iii. the uniform thickness of the layer isregulated by the spacers and the plates, is less than 250 um, and issubstantially less than the linear dimension of the predetermined areaof each storage site; (e) after (d) and while the plates are in theclosed configuration, either. (1) incubating the sample for a relevanttime length and then stopping the incubation; or (2) incubating thesample for a time that is equal or longer than the minimum of a relevanttime length and then assessing, within a time period that is equal orless than the maximum of the relevant length of time, the binding ofeach target analyte to a binding site; wherein the relevant time lengthis: i. equal to or longer than the time that it takes for a targetanalyte of (a) to diffuse across the thickness of the uniform thicknesslayer at the closed configuration; and ii. significantly shorter thanthe time that it takes a target analyte of (a) to laterally diffuseacross the smallest linear dimension of the predetermined area of astorage site or binding site; thereby producing a reaction in which, atthe end of the incubation in (1) or during the assessing in (2), themajority of the capture agent-target analyte-detection agent sandwichbound to each binding site is from a corresponding relevant volume ofthe sample; wherein the incubation allows each target analyte to bind toa binding site and a detection agent, wherein the corresponding relevantvolume is a portion of the sample that is above the correspondingstorage site at the closed configuration, wherein the separation betweenthe edges of the neighboring storage sites and the separation betweenthe edges of the neighboring binding sites are larger than the distancethat a target analyte or detection agent can diffuse in the relevanttime, and wherein there is no fluidic isolation between the binding sitesites and/or the storage sites.

Any embodiment of the multiplex assay device described above may be usedin this method.

13 Quantification by Correcting Effects Generated by None-Sample Volume(C)

In a CROF process, often a sample is mixed with a none-sample-volume(s)which is due to objects that are not the sample, that include, but notlimited to, spacers, air bubbles, dusts, or any combinations of thereof.The air bubbles or dust can be introduced using the sample deposition orother process in the CROF process. These none-sample objects occupyvolume and inside the sample, which should be corrected in determine arelevant volume (a volume of interest) of a sample. One aspect of thepresent invention is to correct the effects generated by the none-samplevolume inside a relevant volume of the sample between two plates, wherethe thickness of the relevant volume is regulated by spacers.

C1. A method for correcting the effects generated by a none-samplematerial in determining a relevant volume of a sample between twoplates, comprising:

-   -   (a) obtaining a sample, wherein a relevant volume of the sample        is to be quantified;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one or both of the plates        comprise spacers and the spacers have a predetermined        inter-spacer distance and height, and each of the spacers is        fixed with its respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), bringing the plates into a closed configuration,        wherein, in the closed configuration: the plates are facing each        other, the spacers and the relevant volume of the sample are        between the plates, the thickness of the relevant volume of the        sample is regulated by the plates and the spacers and is thinner        than the maximum thickness of the sample when the plates are in        the open configuration, and the relevant volume may contain a        volume of a none-sample material;    -   (e) measuring, while the plates are in the closed        configuration, (i) the lateral area of the relevant volume of        the sample and (ii) the volume of the none-sample material; and    -   (f) calculating the relevant volume of the sample by using the        thickness of the relevant volume regulated by the spacers and        correcting the effects of a none-sample material;        wherein the relevant volume is at least a portion of an entire        volume of the sample, and the none-sample materials are the        materials that are not from the sample.    -   the measuring of the none-sample volume is by imaging of the        sample between the two plates.

14 Precision Quantification by Double Checking the Spacing

In a CROF, for a given set of conditions, even the spacers and theplates can give a predetermining sample thickness at a closedconfiguration, the actual set of conditions during a particular CROF maybe different from the expected, which lead to errors in thepredetermined final sample thickness. To reduce such errors, one aspectof the present invention is to double check the final sample thicknessat a closed configuration.

C2. A method for determining and checking a thickness of a relevantvolume of a sample between two plates, comprising:

-   -   (a) obtaining a sample, wherein a relevant volume of the sample        is to be quantified;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one or both of the plates        comprise spacers and the spacers have a predetermined        inter-spacer distance and height, and each of the spacers is        fixed with its respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are partially or completely separated apart and the        spacing between the plates is not regulated by the spacers;    -   (d) after (c), bringing the plates into a closed configuration,        wherein, in the closed configuration: the plates are facing each        other, the spacers and the relevant volume of the sample are        between the plates, the thickness of the relevant volume of the        sample is regulated by the plates and the spacers and is thinner        than the maximum thickness of the sample when the plates are in        the open configuration, and the relevant volume may contain a        volume of a none-sample material;    -   (e) measuring, while the plates are in the closed        configuration, (i) the lateral area of the relevant volume of        the sample and (ii) the volume of the none-sample material; and    -   (f) calculating the relevant volume of the sample by correcting        the effects of a none-sample material;        wherein the relevant volume is at least a portion of an entire        volume of the sample, and the none-sample materials are the        materials that are not from the sample.

15 Wash (WS)

In the present invention, one or any combinations of the embodiments ofthe plate pressing and holding described herein are used in all themethods and devices described in the entire description of the presentinvention.

A method for a wash step in assaying, comprising:

-   -   (a) Performing the steps in one or any combination of the        methods described in above and    -   (b) washing away the sample or the transfer media between the        plates.        In the method that uses CROF, the wash is performed by keep the        plates in the closed-configuration.

In the method that uses CROF, the wash is performed by separating theplates from the closed-configuration.

16 Assays with Multiple Steps (MA)

In the present invention, the embodiments descripted by the disclosures(i.e. all sections) can be used in a combined (a) by combining oneembodiment with other embodiment(s), by using the same embodiment(s)more than one times, and (c) any combination of (a) and (b).

MA1. A method for assaying an analyte in a sample, comprising:

-   -   (a) obtaining a sample with an analyte;    -   (b) performing the method that uses CROF; and    -   (c) separating the plates and performing the method that uses        CROF.

In the method of paragraph MA1, in some embodiments, it furthercomprises, after the step (c) of MA1, a step of repeating the same stepsof all the steps in the method of MA1 at least once.

MA2. A method for assaying an analyte in a sample, comprising:

-   -   (a) obtaining a sample with an analyte;    -   (b) performing the method that uses CROF;    -   (c) separating the plates and performing the method (washing)        that uses CROF; and    -   (d) performing the method that uses CROF.

In the method of paragraph MA2, in some embodiments, it furthercomprises, after the step (d) in MA2, a step of repeating the same stepsof all the steps in the method of MA2 at least once.

In the method of paragraph MA2, in some embodiments, it furthercomprises, after the step (c) in MA2, a step of repeating the same stepsof all the steps in the method of MA1 at least once.

MA3. A kit for assaying an analyte in a sample, comprising:

-   -   a first CROF device that uses CROF; and    -   a third plate that, when the plates of the first CROF device are        separated, combines with one of the plates of the first CROF        device to form a second CROF device.        MA4. A kit for assaying an analyte in a sample, comprising:    -   a first CROF device that uses CROF;    -   at least one binding site or storage site that is on the sample        contact area of the plate of a CROF device; and    -   a third plate that, when the plates of the first CROF device are        separated, combines with one of the plates of the first CROF        device to form a second CROF device;        wherein the binding site binds a target analyte to the plate        surface, and the storage site has a reagent that, upon in touch        with the sample, can be dissolved into the sample and diffuse in        the sample.

The imaging may comprise a use of a smart phone. The methods of thissection may further comprise a step of illumination by a light source.The light source may be a laser, LED, a lamp, or a camera flash light.

A Kit (MQXA) for Performing Assay for Detecting a Target Entity in aSample

A kit for assaying a target entity in a sample, may comprise:

-   -   a. a first plate, wherein one surface of the first plate has one        or a plurality of binding site(s) that can immobilize a target        entity and the binding site has binding partner that binds the        target entity;    -   b. a cover plate;    -   c. a sample in the inner space between the cover plate and the        first plate, wherein the sample contains said target entity that        is mobile in the sample, the shape of sample is deformable, the        first plate and the second plate are movable relative to each        other, the shape of the sample is substantially conformal to the        inner surfaces, at least a part of the sample is in contact to        the binding site, and the inner spacing is, during incubation,        less than certain distance. the sample is in contact with said        binding sites;    -   d. an imaging device that can image the first plate surface        and/or the cover plate surface; and    -   e. a measuring device that can measure the spacing of the inner        space.

The methods of this section may include use of a smart phone. Themethods of this section may include use of an illuminating device. Theilluminating device may comprise a laser, LED, a lamp, or a camera flashlight.

17 Plate Pressing and Holding (H) Compressing Forces.

In a CROF process, forces are used to compress the two plates to bringthe plates from an open configuration to a closed configuration. Thecompressing forces reduce the spacing between the inner surfaces of theplates and hence a thickness of the sample that is between the plates.In the present invention, the compressing forces include, but notlimited to, mechanical force, capillary forces (due to surfacetensions), electrostatic force, electromagnetic force (including thelight), and any combination of thereof.

In some embodiments of bring the plates from an open configuration to aclosed configuration, an external force is applied to push the firstplate and the second plate to toward each other.

In some embodiments of bring the plates from an open configuration to aclosed configuration, an external pressure is applied to outside thefirst plate and the second plate to push the plates toward each other,and the pressure is higher than the pressure inside of the plate. Adevice is used to make the pressure of outside the plates higher thanthat inside the plate. The device include, in limited to, a sealingdevice.

In some embodiments, the compress force is at least partially providedby the capillary force, which is due to a liquid between the first plateand the second plate and the corresponding surface tensions andinteractions with the plates. In some embodiments, the liquid is thesample itself, or the sample mixed with liquid. In certain embodiments,capillary force is used together with other forces. In many cases, asample is often in liquid and the surface tensions are suited forinserting a capillary force. In some embodiments, the sample deformationby the plates can automatically stop when the capillary force equals tothe force needed to deform the sample.

In certain embodiments, the compressing force (hence the sampledeformation) is created by isolating the pressure between the firstplate and the second plate (inside pressure) from that outside of theplates (outside pressure), and then make the inside pressure lower thanthe outside pressure. The isolation can be done using a vacuum seal orother devices.

In some embodiments, it is a combination of the methods described above.

Gradual Pressing.

In certain embodiments, the compressing force to bring the plates to aclosed configuration is applied in a process, termed “gradual pressing”,which comprises: pressing (i.e. applying the compressing the force) isapplied at one location of the plate(s) first, then is applied graduallyto other locations of the sample. In some embodiments of the gradualpressing, the compressing force (except the capillary forces by thesample itself) at one location is, after deformed the sample to adesired thickness at that location, (i) maintained during the entireprocess of the pressing and the sample deformation, (ii) removed whileother locations being pressed, or (iii) a use of (i) for certain part ofthe plates and a use of (ii) for other part of the sample.

In one embodiment of the gradual pressing, a roller is being used topress the first plate and the second plate (the sample is between theplates, and the plates are slightly flexible) against another roller ora flat surface.

In another embodiment, the human fingers are the tool of the pressingthe plates (hence the sample). The pressing is one part of human handagainst another part of human body (including another part of humanhand) or a human hand against an object (e.g. a table surface).

In one embodiment, the pressing starts at one location of the sample andgradual moved to other locations of the sample.

In one embodiment of the gradual pressing, a pressed air jet is firstdirected to a location (e.g. the center) of the plate pair (which isbetween the first plate and the second plate, one of the plates isslightly flexible) and the pressure is gradually extended to other partof the plate pair.

In another embodiment, one or both of the first plate and the secondplate is flexible and is in contact with one location of the sample,then a capillary force in that location pulls the plate pair together(toward to each other) to deform the sample.

Advantage of the gradual pressing include: it allows one to use lessforce to deform the sample (because for the same force, the smallerpress area, the larger the pressure); it helps motion (deformation) ofthe sample, and/or it reduces air bubble in the sample. The largerpressure is, the more sample deformation will be. A gradual pressing canimprove the thickness uniformity of the deformed sample.

Pressing Devices.

The devices for asserting the compressing force(s) for the sampledeformation in CROF have several implementations. Some embodiments areto use human hand to press, for example, to press by human fingers.Certain embodiments are to use a press device, where the press deviceincludes, but not limited to, a human hand(s), a mechanical clip, amechanical press, mechanical clamp, a mechanical slider, a mechanicaldevice, ab electromagnetic device, roller that rolls on a surface, tworollers against each other, fluidic press, a hydraulic device, or anycombination of thereof. Certain embodiments are use pressured liquid(including pressed air) to press the first plate and/or the second platedirectly or indirectly. “Directly” means the pressured liquid is applieddirectly on the first plate and/or the second plate; and the“indirectly” means it is applied through a third object. Certainembodiments in pressing use a combination of the above embodiments ofpressing devices and methods.

Furthermore, in some embodiments of the sample deformation, the pressingand the sample deformation are monitored. The monitoring can be used tocontrol the pressing and the sample deformation. The monitoring of thedeformation include, but not limited to, a mechanical method,electrical, optical, chemical, magnetic, and any combination of thereof.The mechanical methods include, but not limited to, mechanical gauges,spacer (mechanical stoppers, more discussed below), and sound waves.

In CROF, the spacing control device comprises mechanical press,mechanical translation stages, human fingers, liquid that providecapillary forces that pulls the plates toward each other, liquid(including air) that applies a pressure on the plates, or a combinationof thereof.

In certain embodiments, the mechanical stages (translational and/orrotational) are used for the sample deformation and sample thicknesscontrol and work together with the monitoring systems.

In some embodiments, the compressing force is at least partly suppliedby a press (which is a device that bring the plates to a closedconfiguration) configured to press the plates together into the closedconfiguration.

In some embodiments, the plate pressing is to use a human hand. Thehuman can be the person being tested or a person who perform the test,or a person who collecting the sample.

In some embodiments, the plate pressing is to hold the two platestogether is to use a capillary force. The capillary force is generatedby making at least a portion of the inner surface of one plate or bothhydrophilic. With a proper capillary force, the two plates is able tomaintain the same plate-spacing and the same thickness of the relevantvolume of the sample as that when the plates initially in the closedconfiguration, even a part or all of the forces (except the capillaryforce) that were used to compress the plate to the close configurationis removed.

In some embodiments, the device that applies a compressing force on theouter surface of the plates to reducing the plate inner surface spacingcomprise a contacting surface that is comfortable to the outer surfacesof the plate, wherein the contacting surface of the device is thesurface of the device that contacts the outer surface of the plates, andthe “conformable to the outer surface of the plate” means that thedevice surface can deform, during the compressing, it shape to conformthe shape of the plate outer surface. In one exemplary embodiment, thecompressing device is human figures. In another exemplary embodiment,the compressing device has a contacting surface made of soft plastics orrubbers.

Self-Holding (Maintaining the Final Sample Thickness after RemovingCompressing Forces).

In some embodiments of pressing in CROF, after the sample deformation ata closed configuration, some of the compressing forces are removed andthe sample maintains the same final sample thickness as the compressionforces still exist. Such situation is termed “self-holding”.

One reason for self-holding is that after removing the compressingforces that were inserted from outside of the plate pair, there arestill other forces exist between the inner surfaces of the plates, suchas a capillary force, which hold the plate pair together. The capillaryforce is the due to the wetting properties of the sample on the plates.

To have self-holding, one needs to control the plate surface wettingproperties, the total contact area of the sample to the plates, thefinal sample thickness at a closed configuration, or a combination ofthereof.

In some embodiments to achieve self-holding, one or both inner surfacesof the plates is hydrophilic. Namely, it is either one of plates have aninner surface that is hydrophilic or both of the plates have an innersurface that is hydrophilic.

The capillary force depends on the radius curvature of the liquidsurface, smaller the curvature and higher the capillary force. A smallercurvature can be achieved by using smaller spacing between the twoplates (i.e. plate pair) and hence a smaller sample thickness. In someembodiments, a final sample thickness for achieving self-holding is 10nm or less, 100 nm or less, 100 nm or less, 500 nm or less, 1 um(micrometer) or less, 2 um or less, 3 um or less, 5 um or less, 10 um orless, 20 um or less, 50 um or less, 70 um or less, 100 um or less, 150um or less, 300 um or less, 500 um or less, 700 um or less, 1000 um orless, 1200 um or less, or a range between any two of the values.

In some embodiments, the area of the sample in contract with the platesfor self-holding is at most 10 um², at most 100 um², at most 200 um², atmost 500 um², at most 1000 um², at most 2000 um², at most 5000 um², atmost 8,000 um², at most 0.01 mm², at most 0.05 mm², at most 0.1 mm², atmost 0.5 mm², at most 1 mm², at most 5 mm2, at most 10 mm², at most 50mm², at most 100 mm², at most 500 mm², at most 1,000 mm², at most 2,000mm², at most 5,000 mm², at most 10,000 mm², at most 100,000 mm², or arange between any two of the values.

In some embodiments, one or both of the plate inner surface's wettingproperties is modified for better self-holding.

HS.1 In some embodiments, in a CROF process, a device is used to inserta compressing force to bring the plates into a closed configuration, andafter the closed configuration is reached, the compressing force by thedevice is removed and the sample thickness and the inner surface spacingof the plates are remained approximately the same as that beforeremoving the compressing force by the device. In some embodiments, inthe methods of previous paragraph, it further comprises a step ofreading a signal from the plates or between the plates, wherein thesignal includes, but not limited to, a signal related to analytes,entity, labels, sample volume, concentration of a matter (i.e.chemicals), or any combination of thereof.

In the method of paragraph SH.1, the device is a human hand(s), amechanical clip, a mechanical press, mechanical clamp, a mechanicalslider, a mechanical device, ab electromagnetic device, roller thatrolls on a surface, two rollers against each other, fluidic press, ahydraulic device, or any combination of thereof.

In the method of paragraph SH.1, in some embodiments, “the samplethickness and the inner surface spacing of the plates are remainedapproximately the same as that before removing the compressing force bythe device” means that the relative difference of the sample thicknessand the plate inner surface spacing before and after removing thecompressing force is 0.001% or less, 0.01% or less, 0.1% or less; 0.5%or less, 1% or less, 2% or less, 5% or less, 8% or less, 10% or less,15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% orless, 70% or less, 80% or less, 90% or less, 99.9% or less, or a rangebetween any of the values.

In the method of paragraph SH.1, in some embodiments, the samplethickness and the inner surface spacing of the plates after removing thecompressing force by the device care predetermined, whereinpredetermined means that the thickness and the spacing after removingthe compressing force is known before applying the compressing force fora given compressing conditions.

H1. A method for reducing the thickness of a relevant volume of a sampleand maintain the reduced thickness, comprising:

-   -   (a) obtaining a sample, wherein a thickness of a relevant volume        of the sample is to be reduced;    -   (b) obtaining two plates that are movable relative to each other        into different configurations, wherein one or both of the plates        comprise spacers and the spacers have a predetermined        inter-spacer distance and height, and each of the spacers is        fixed with its respective plate;    -   (c) depositing, when the plates are configured in an open        configuration, the sample on one or both of the plates; wherein        the open configuration is a configuration in which the two        plates are either partially or completely separated apart and        the spacing between the plates is not regulated by the spacers;    -   (d) after (c), spreading the sample by using a pressing device        that brings the plates into a closed configuration, wherein, in        the closed configuration: the plates are facing each other, the        spacers and the relevant volume of the sample are between the        plates, the thickness of the relevant volume of the sample is        regulated by the plates and the spacers and is thinner than the        maximum thickness of the sample when the plates are in the open        configuration, and at least one of the spacers is inside the        sample; and    -   (e) after (d), releasing the device, wherein after releasing the        pressing device, the spacing between the plates remains the same        as or approximately same as that when the device is applied.        -   wherein the relevant volume is at least a portion of an            entire volume of the sample.

In the method of paragraph H1, the approximately same as the spacingbetween the plates is at most 1%, at most 2%, at most 5%, at most 10%,at most 20%, at most 50%, at most 60%, at most 70%, at most 80%, at most90%, or a range between any two of the values.

For example, in CROF, a human hand or hands are used to compressed twoplate to a closed position, then the hand(s) and hence the compressingforce by hand(s) are removed, but the final sample thickness is stillthe same as that when the compressing force by hands exist.

18 Other Combinations

In the present invention, each of the embodiments in the disclosures(i.e. all sections) can be used (a) alone, (b) combined with otherembodiment(s), (c) multiple times, and (d) any combination of (a) to(c).

The methods and devices in the present invention disclosed can be usedalone or any combination of thereof. The term a “QMAX” method or devicerefers to a method or device of the embodiments described here.

In some embodiments, the methods and devices in the present inventiondisclosed can be used in the form of Q, X, A, M, QX, QA, QM, XA, XM, AM,QXA, QAM, XAM, and QXAM.

Some embodiments of application of the Q, X, A, and M to surfaceimmobilization assay, comprising

-   -   a. having a first plate, wherein the first plate surface has at        least one well of a known depth and volume, and bottom surface        of the well has one or a plurality of binding site(s) that can        immobilize a target entity in a sample;    -   b. depositing, into the well, the sample of a volume        approximately the same as the well volume, wherein the sample        contains the targeted entity, the targeted entity is mobile in        the sample, the shape of sample is deformable, and the sample        covers only a part of the well (hence have a simple thickness        higher than the well depth);    -   c. having a cover plate;    -   d. facing the first plate and the cover plate to each other,        wherein the sample is between the inner surfaces of the first        plate and the second plate;    -   e. reducing the sample thickness by reducing the spacing between        the inner surfaces of the first plate and the second plate; and    -   f. Incubating the sample at the reduced sample thickness for a        period of time;

One variation of these methods is to apply one or more of the abovesteps to 96 well plates or other well plates.

The methods and devices in the present invention disclosed in Section 1,2, 3, and 5, can be used alone or any combination of thereof.Specifically, we use Q for the inventions disclosed in Section 1 and 2,A for the inventions disclosed in Section 3 and 5, X for the inventionsdisclosed in Section 4 and 5, and M for the inventions disclosed inSection 6. Hence the methods and devices in the present inventiondisclosed in Section 1, 2, 3, and 5, can be used in the form of Q, X, A,M, QX, QA, QM, XA, XM, AM, QXA, QAM, XAM, and QXAM.

Some embodiments of application of the Q, X, A, and M to surfaceimmobilization assay, comprising

-   -   a. having a first plate, wherein the first plate surface has at        least one well of a known depth and volume, and bottom surface        of the well has one or a plurality of binding site(s) that can        immobilize a target entity in a sample;    -   b. depositing, into the well, the sample of a volume        approximately the same as the well volume, wherein the sample        contains the targeted entity, the targeted entity is mobile in        the sample, the shape of sample is deformable, and the sample        covers only a part of the well (hence have a simple thickness        higher than the well depth);    -   c. having a cover plate;    -   d. facing the first plate and the cover plate to each other,        wherein the sample is between the inner surfaces of the first        plate and the second plate;    -   e. reducing the sample thickness by reducing the spacing between        the inner surfaces of the first plate and the second plate; and    -   f. Incubating the sample at the reduced sample thickness for a        period of time.

One variation of these methods is to apply one or more of the abovesteps to 96 well plates or other well plates.

Several embodiments of the methods, devices, and systems combine one ormore of the features of sample volume quantification (Q), reagentsaddition (A), and/or assay acceleration (X) (and may be referred to asthe corresponding acronyms QA, QX, AX, and QAX). Some experimentaldemonstrations of Q, A, X, QA, QX, AX, and QAX methods and devices aredescribed below.

19 Reagents

The term “reagents” refers to, unless stated otherwise, one or more ofbiological agents, biochemical agents, and/or chemical agents. Forexample, reagents may include capture agents, detection agents, chemicalcompounds, optical labels, radioactive labels, enzymes, antibodies,proteins, nucleic acids, DNA, RNA, lipids, carbohydrates, salts, metals,surfactants, solvents, or any combination of thereof.

In some embodiments, the reagents on a plate in the form of liquid,solid, molecular vapor, or a combination of thereof. The deposition ofreagent, include, but are not limited to, depositing, placing, printing,stamping, liquid dispensing, evaporation (thermal evaporation, vaporevaporation, human breathing), chemical vapor deposition, and/orsputtering. Different reagents can be in different locations. Reagentsmay be printed and/or deposited as small dots of reagents.

In some embodiments, the reagents are deposited on a plate in a liquidor vapor form first, then are dried to become dry reagents on the platebefore a CROF process.

Controlling Reagents Releasing Time.

A-methods may further comprise a step of controlling the reagent releasetime (i.e. the time measures how fast a reagent can be dissolved in asample. Some embodiments in controlling the reagent release time of areagent comprises a step of mixing or coating on top of the reagent a orseveral “releasing control material(s)” that affect the release (intothe sample) of the reagent. In some embodiments, the releasing controlmaterial can be another reagent. For example, there are two reagents Aand B, the reagent A is coated on top of the reagent B, under certainconditions, the reagent A will be dissolved into the sample before thereagent B.

Furthermore, the surface properties of the first plate and the secondplate may be used to control the reagent release. One example is tocontrol the surface wetting properties. For many reagents, a hydrophobicsurface binds the reagent well, hence leading to slow release or norelease of the reagent into the sample (depending upon how thick is thereagent layer), while a hydrophilic surface binds the reagent poorlyhence leading a fast release into the sample.

Drying of Reagents.

In some embodiments, after the reagent deposition step (c) but beforethe sample deposition step (d), A-methods further comprise a step ofdrying some or all of the reagents deposited in the step (c).

Location of Reagents.

Reagents may be applied and/or arranged on one or both of the plates.Reagents may be in storage sites (locations) on the plate(s), with eachstorage site including one or more reagents. Different storage sites mayinclude different reagents, the same reagents, or one or more commonreagents.

Control Concentration of Added Reagents. In some embodiments, themethods may further comprise a step of controlling the concentration ofthe added reagents by controlling the samples thickness over the storagesites (i.e., the surface with reagents).

The reagent used in the present invention may be any suitable reagentrequired for an assay, e.g., a labeled or unlabeled antibody, a labeledor unlabeled nucleic acid, an enzyme that may or may not contain anaffinity moiety, etc. In some embodiments and as noted above, the storedreagent may be a component of an assay designed to test a blood or otherliquid sample for the presence of an analyte. For example, choride ionscan be measured by any of the following protocols, and components ofthese assays may be present in a storage site: Colorimetric methods:chloride ions displace thiocyanate from mercuric thiocyanate. Freethiocyanate reacts with ferric ions to form a colored complex-ferricthiocyanate, which is measured photometrically. Coulometric methods:passage of a constant direct current between silver electrodes producessilver ions, which react with chloride, forming silver chloride. Afterall the chloride combines with silver ions, free silver ions accumulate,causing an increase in current across the electrodes and indicating theend point to the reaction. Mercurimetric methods: chloride is titratedwith a standard solution of mercuric ions and forms HgCl2 solublecomplex. The end point for the reaction is detected colorimetricallywhen excess mercury ions combine with an indicator dye,diphenylcarbazon, to form a blue color. Likewise, magnesium can bemeasured colorimetrically using calmagite, which turns a red-violetcolor upon reaction with magnesium; by a formazan dye test; emits at 600nm upon reaction with magnesium or using methyithymol blue, which bindswith magnesium to form a blue colored complex. Likewise, calcium can bedetected by a colorimetric technique using O-Cresolphtalein, which turnsa violet color upon reaction of O-Cresolphtalein complexone withcalcium. Likewise, Bicarbonate cab ne tested bichromatically becausebicarbonate (HCO3⁻) and phosphoenolpyruvate (PEP) are converted tooxaloacetate and phosphate in the reaction catalyzed byphosphoenolpyruvate carboxylase (PEPC). Malate dehydrogenase (MD)catalyzes the reduction of oxaloacetate to malate with the concomitantoxidation of reduced nicotinamide adenine dinucleotide (NADH). Thisoxidation of NADH results in a decrease in absorbance of the reactionmixture measured bichromatically at 380/410 nm proportional to theBicarbonate content of the sample. Blood urea nitrogen can be detectedin a colorimetric test in which diacetyl, or fearon develops a yellowchromogen with urea and can be quantified by photometry, or multiusingthe enzyme urease, which converts urea to ammonia and carbonic acid,which can be assayed by, e.g., i) decrease in absorbance at 340 nm whenthe ammonia reacts with alpha-ketoglutaric acid, ii) measuring the rateof increase in conductivity of the solution in which urea is hydrolyzed.Likewise, creatinine can be measured colorimetrically, by treated thesample with alkaline picrate solution to yield a red complex. Inaddition, creatine can be measured using a non-Jaffe reaction thatmeasures ammonia generated when creatinine is hydrolyzed by creatinineiminohydrolase. Glucose can be measured in an assay in which blood isexposed to a fixed quantity of glucose oxidase for a finite period oftime to estimate concentration. After the specified time, excess bloodis removed and the color is allowed to develop, which is used toestimate glucose concentration. For example, glucose oxidase reactionwith glucose forms nascent oxygen, which converts potassium iodide (inthe filter paper) to iodine, forming a brown color. The concentration ofglycosylated hemoglobin as an indirect read of the level of glucose inthe blood. When hemolysates of red cells are chromatographed, three ormore small peaks named hemoglobin Ala, Alb, and Alc are eluted beforethe main hemoglobin A peak. These “fast” hemoglobins are formed by theirreversible attachment of glucose to the hemoglobin in a two-stepreaction. Hexokinase can be measured in an assay in which glucose isphosphorylated by hexokinase (HK) in the presence of adenosinetriphosphate (ATP) and magnesium ions to produce glucose-6-phosphate andadenosine diphosphate (ADP). Glucose-6-phosphate dehydrogenase (G6P-DH)specifically oxidises glucose-6-phosphate to gluconate-6-phosphate withthe concurrent reduction of NAD+ to NADH. The increase in absorbance at340 nm is proportional to the glucose concentration in the sample. HDL,LDL, triglycerides can be measured using the Abell-Kendall protocol thatinvolves color development with Liebermann-Burchard reagent (mixedreagent of acetic anhydride, glacial acetic acid, and concentratedsulfuric acid) at 620 nm after hydrolysis and extraction of cholesterol.A fluorometric analysis may be used utilized to determine triglyceridereference values. Plasma high-density lipoprotein cholesterol (HDL-C)determination is measured by the same procedures used for plasma totalcholesterol, after precipitation of apoprotein B-containing lipoproteinsin whole plasma (LDL and VLDL) by heparin-manganese chloride. Thesecompounds can also be detected colorimetrically in an assay that isbased on the enzyme driven reaction that quantifies both cholesterolesters and free cholesterol. Cholesterol esters are hydrolyzed viacholesterol esterase into cholesterol, which is then oxidized bycholesterol oxidase into the ketone cholest-4-en-3-one plus hydrogenperoxide. The hydrogen peroxide is then detected with a highly specificcolorimetric probe. Horseradish peroxidase catalyzes the reactionbetween the probe and hydrogen peroxide, which bind in a 1:1 ratio.Samples may be compared to a known concentration of cholesterolstandard.

Data Processing.

In certain embodiments, the subject device is configured to process dataderived from reading the CROF device. The device may be configured inany suitable way to process the data for use in the subject methods. Incertain embodiments, the device has a memory location to store the dataand/or store instructions for processing the data and/or store adatabase. The data may be stored in memory in any suitable format.

In certain embodiments, the device has a processor to process the data.In certain embodiments, the instructions for processing the data may bestored in the processor, or may be stored in a separate memory location.In some embodiments, the device may contain a software to implement theprocessing.

In certain embodiments, a device configured to process data acquiredfrom the CROF device contains software implemented methods to performthe processing. Software implemented methods may include one or more of:image acquisition algorithms; image processing algorithms; userinterface methods that facilitate interaction between user andcomputational device and serves as means for data collection,transmission and analysis, communication protocols; and data processingalgorithms. In certain embodiments, image processing algorithms includeone or more of: a particle count, a LUT (look up table) filter, aparticle filter, a pattern recognition, a morphological determination, ahistogram, a line profile, a topographical representation, a binaryconversion, or a color matching profile.

In certain embodiments, the device is configured to display informationon a video display or touchscreen display when a display page isinterpreted by software residing in memory of the device. The displaypages described herein may be created using any suitable softwarelanguage such as, for example, the hypertext markup language (“HTML”),the dynamic hypertext markup language (“DHTML”), the extensiblehypertext markup language (“XHTML”), the extensible markup language(“XML”), or another software language that may be used to create acomputer file displayable on a video or other display in a mannerperceivable by a user. Any computer readable media with logic, code,data, instructions, may be used to implement any software or steps ormethodology. Where a network comprises the Internet, a display page maycomprise a webpage of a suitable type.

A display page according to the invention may include embedded functionscomprising software programs stored on a memory device, such as, forexample, VBScript routines, JScript routines, JavaScript routines, Javaapplets, ActiveX components, ASP.NET, AJAX, Flash applets, Silverlightapplets, or AIR routines.

A display page may comprise well known features of graphical userinterface technology, such as, for example, frames, windows, scrollbars, buttons, icons, and hyperlinks, and well known features such as a“point and click” interface or a touchscreen interface. Pointing to andclicking on a graphical user interface button, icon, menu option, orhyperlink also is known as “selecting” the button, option, or hyperlink.A display page according to the invention also may incorporatemultimedia features, multi-touch, pixel sense, IR LED based surfaces,vision-based interactions with or without cameras.

A user interface may be displayed on a video display and/or displaypage. The user interface may display a report generated based onanalyzed data relating to the sample, as described further below.

The processor may be configured to process the data in any suitable wayfor use in the subject methods. The data is processed, for example, intobinned data, transformed data (e.g., time domain data transformed byFourier Transform to frequency domain), or may be combined with otherdata. The processing may put the data into a desired form, and mayinvolve modifying the format of data. Processing may include detectionof a signal from a sample, correcting raw data based on mathematicalmanipulation or correction and/or calibrations specific for the deviceor reagents used to examine the sample; calculation of a value, e.g., aconcentration value, comparison (e.g., with a baseline, threshold,standard curve, historical data, or data from other sensors), adetermination of whether or not a test is accurate, highlighting valuesor results that are outliers or may be a cause for concern (e.g., aboveor below a normal or acceptable range, or indicative of an abnormalcondition), or combinations of results which, together, may indicate thepresence of an abnormal condition, curve-fitting, use of data as thebasis of mathematical or other analytical reasoning (includingdeductive, inductive, Bayesian, or other reasoning), and other suitableforms of processing. In certain embodiments, processing may involvecomparing the processed data with a database stored in the device toretrieve instructions for a course of action to be performed by thesubject.

In certain embodiments, the device may be configured to process theinput data by comparing the input data with a database stored in amemory to retrieve instructions for a course of action to be performedby the subject. In some embodiments, the database may contain storedinformation that includes a threshold value for the analyte of interest.The threshold value may be useful for determining the presence orconcentration of the one or more analytes. The threshold value may beuseful for detecting situations where an alert may be useful. The datastorage unit may include records or other information that may be usefulfor generating a report relating to the sample.

In certain embodiments, the device may be configured to receive datathat is derived from the CROF device. Thus in certain cases, the devicemay be configured to receive data that is not related to the sampleprovided by the subject but may still be relevant to the diagnosis. Suchdata include, but are not limited to the age, sex, height, weight,individual and/or family medical history, etc. In certain embodiments,the device is configured to process data derived from or independentlyfrom a sample applied to the CROF device.

20 Packages

Another aspect of the present invention is related to packaging, whichwould prolong the lifetime of the reagent used and facilitate the easyof the use.

In some embodiments, the plates in CROF with or without reagents are putinside a package, either one plate per package or more than one platesper package. In one embodiment, the first plate and second plate arepackaged in a different package before a use. In some embodiments,different assays share a common first plate or a common second plate.

In some embodiments, each of the packages is sealed. In someembodiments, the seal is for preventing the air, chemicals, moisture,contamination, or any combination of them from outside of the packagefrom entering inside the package. In some embodiments, the package isvacuum sealed or fill with nitrogen gas, or inner gases. In someembodiments, a material that can prolong a shelf-life-time of the plateand/or the reagents (including the capture agents, detection agents,etc.) is packaged inside the package with a plate.

In some embodiments, the package materials are a thin layer form, sothat the package can be easily tom apart by a human hand.

21. Homogenous Assay Using a Signal Amplification Surface

In many applications of an assay, particularly in PoC or other fastassays, it is desirable to avoid washing steps. One aspect of thepresent invention is related to the devices, systems, and methods thatcan avoid washing of the assay.

By incorporating and/or using a signal amplification surface, thedisclosed devices, systems, and methods may facilitate performing assayswithout washing. The surface amplification surface may only amplify thelight emitted in a small distance from the surface (e.g. 20 nm, or 50nm, or 100 nm). One example of the surface amplification layer is D2PA.

It is believed that the following claims particularly point out certaincombinations and subcombinations that are directed to one of thedisclosed inventions and are novel and non-obvious. Inventions embodiedin other combinations and subcombinations of features, functions,elements and/or properties may be claimed through amendment of thepresent claims or presentation of new claims in this or a relatedapplication. Such amended or new claims, whether they are directed to adifferent invention or directed to the same invention, whetherdifferent, broader, narrower, or equal in scope to the original claims,are also regarded as included within the subject matter of theinventions of the present disclosure.

What is claimed is:
 1. A device for collecting and analyzing a vaporcondensate (VC) sample, comprising: a collection plate and a coverplate, wherein: i. the plates are movable relative to each other intodifferent configurations; ii. one or both plates are flexible; iii. eachof the plates has, on its respective surface, a sample contact area forcontacting a vapor condensate (VC) sample that contains an analyte; iv.one or both of the plates comprise spacers, wherein at least one of thespacers is inside the sample contact area; wherein one of theconfigurations is an open configuration, in which: the two plates areeither completely or partially separated apart, the spacing between theplates is not regulated by the spacers, and the VC sample is depositedon one or both of the plates by directly condensing a vapor on the plateor the plates; and wherein another of the configurations is a closedconfiguration which is configured after the VC sample deposition in theopen configuration; and in the closed configuration: at least a part ofthe VC sample is between the two plates and in contact with the twoplates, and has a highly uniform thickness that is regulated by thespacers and the two sample surfaces of the plates and is equal to orless than 30 um with a small variation.
 2. The device of claim 1,wherein the device further comprises a dry reagent coated on one or bothof the plates.
 3. The device of claim 1, wherein the device furthercomprises, on the one or both plates, a dry binding site that has apredetermined area, wherein the dry binding site binds to andimmobilizes the analyte in the sample.
 4. The device of claim 1, whereinthe sample is an exhale breath condensate, wherein the exhale breathcondensate directly condenses on one or both the sample contactingareas.
 5. The device of claim 1, wherein the sample is a vapor from abiological sample, an environmental sample, a chemical sample, or aclinical sample.
 6. The device of claim 1, wherein the analyte comprisesa molecule (e.g., a protein, peptides, DNA, RNA, nucleic acid, or othermolecules), cells, tissues, viruses, or nanoparticles with differentshapes.
 7. The device of claim 1, wherein the analyte comprises avolatile organic compound (VOCs).
 8. The device of claim 1, wherein theanalyte comprises nitrogen, oxygen, CO₂, H₂O, or inert gases.
 9. Thedevice of claim 1, wherein the analyte is stained.
 10. The device ofclaim 1, wherein the highly uniform thickness has a value (i) equal toor less than 0.5 um, (ii) in the range of 0.5 um to 1 um, (iii) in therange of 1 um to 2 um, or (iv) in the range of 2 um to 10 um.
 11. Thedevice of claim 1, wherein a material of the plate and the spacers areindependently selected from the group consisting of polystyrene, PMMA,PC, COC, COP, and another plastic.
 12. The device of claim 1, whereinthe inter-spacer distance is (i) in the range of 1 um to 200 um, or (ii)in the range of 200 um to 1000 um.
 13. The device of claim 1, whereinthe VC sample is an exhaled breath condensate from a human or an animal.14. The device of claim 1, wherein the spacers regulating the layer ofuniform thickness have a filling factor of at least 1%, wherein thefilling factor is a ratio of a spacer area in contact with the layer ofuniform thickness to a total plate area in contact with the layer ofuniform thickness.
 15. The device of claim 1, wherein for spacersregulating the layer of uniform thickness, a Young's modulus of thespacers times a filling factor of the spacers is equal to or larger than10 MPa, wherein the filling factor is the ratio of a spacer area incontact with the layer of uniform thickness to a total plate area incontact with the layer of uniform thickness.
 16. The device of claim 1,wherein for a flexible plate, a thickness of the flexible plate times aYoung's modulus of the flexible plate is in the range of 60 to 750GPa-um.
 17. The device of claim 1, wherein for a flexible plate, afourth power of the inter-spacer distance (ISD) divided by a thicknessof the flexible plate (h) and a Young's modulus (E) of the flexibleplate, ISD⁴/(hE), is equal to or less than 10⁶ um³/GPa.
 18. The deviceof claim 1, wherein the layer of uniform thickness of the sample isuniform over a lateral area that is at least 1 mm².
 19. The device ofclaim 1, wherein the device is configured to analyze the sample in 60seconds or less.
 20. The device of claim 1, wherein the device furthercomprises, on one or both of the plates, one or a plurality ofamplification sites that are each capable of amplifying a signal fromthe analyte or a label of the analyte when the analyte or label iswithin 500 nm from the amplification site.
 21. The device of claim 1,wherein the device further comprises a detector, wherein the detector is(i) an optical detector for detecting an optical signal, or (ii) anelectrical detector for detecting an electrical signal.
 22. A system forrapidly analyzing a vapor condensation sample using a mobilecommunication device comprising: (a) a device of claim 1; and (b) amobile communication device comprising: i. one or a plurality of camerasfor detecting a signal and/or imaging the vapor condensate sample; andii. electronics, signal processors, hardware and software for receivingand/or processing the detected signal and/or the image of the vaporcondensate sample and for remote communication.
 23. A method foranalyzing an analyte in a vapor condensate sample, comprising: obtaininga device of claim 1; depositing a vapor condensate (VC) sample onto oneor both plates of the device, wherein the deposition is by condensingthe vapor directly on the one or both plates; bringing the two platesinto a closed configuration; and analyzing the analyte in the layer ofuniform thickness while the plates are in the closed configuration. 24.A method for analyzing an analyte in a vapor condensate sample, whereinthe method comprises: (a) obtaining a sample; (b) obtaining the deviceof claim 1 wherein the spacers have: i. a pillar shape withsubstantially uniform cross-section and a flat top surface; ii. a ratioof the width to the height equal to or larger than one; iii. apredetermined constant inter-spacer distance that is in the range of 10um to 200 um; and iv. a filling factor equal to 1% or larger; and (c)depositing the sample on one or both of the plates when the plates areconfigured in the open configuration; (d), after (c), using the twoplates to compress at least part of the sample into the layer ofsubstantially uniform thickness that has an average value equal to orless than 30 um with a variation of less than 10%, wherein thecompressing comprises: bringing the two plates together; and conformablepressing, either in parallel or sequentially, an area of at least one ofthe plates to press the plates together to a closed configuration,wherein the conformable pressing generates a substantially uniformpressure on the plates over the at least part of the sample, and thepressing spreads the at least part of the sample laterally between thesample contact surfaces of the plates, and wherein the closedconfiguration is a configuration in which the spacing between the platesin the layer of uniform thickness region is regulated by the spacers;and (e) analyzing the analyte in the layer of uniform thickness whilethe plates are the closed configuration; wherein the filling factor isthe ratio of the spacer contact area to the total plate area; wherein aconformable pressing is a method that makes the pressure applied over anarea is substantially constant regardless the shape variation of theouter surfaces of the plates; and wherein the parallel pressing appliesthe pressures on the intended area at the same time, and a sequentialpressing applies the pressure on a part of the intended area andgradually move to other area.
 25. The method of claim 23, furthercomprising removing the external force after the plates are in theclosed configuration; imaging the analytes in the layer of uniformthickness while the plates are the closed configuration; and counting anumber of analytes or the labels in an area of the image.
 26. The methodof claim 24, wherein the analyzing step comprises counting the analytein the sample, and wherein an imaging and the counting is done by: i.illuminating the cells in the layer of uniform thickness; ii. taking oneor more images of the cells using a CCD or CMOS sensor; iii. identifyingcells in the image using a computer; and iv. counting a number of cellsin an area of the image.
 27. The method of claim 23, wherein theexternal force is provided by a human hand.
 28. The method of claim 24,wherein the device further comprises a dry reagent that is coated on oneor both plates.
 29. The method of claim 24, wherein the analytecomprises a molecule (e.g., a protein, peptides, DNA, RNA, nucleic acid,or other molecules), cells, tissues, viruses, nanoparticles withdifferent shapes, volatile organic compounds (VOCs), nitrogen, oxygen,CO2, H2O, or inert gases.
 30. The method of claim 24, wherein thesubstantially uniform thickness has a value between 0.5 um to 10 um.